Synthetic DNA vectors and methods of use

ABSTRACT

Provided herein are isolated DNA vectors comprising a heterologous gene, wherein the DNA vector is devoid of bacterial plasmid DNA and/or bacterial signatures, which can abrogate persistence in vivo. The invention also features pharmaceutical compositions (non-immunogenic pharmaceutical compositions) including the DNA vectors of the invention, which can be used for induction of long-term, episomal expression of a heterologous gene in a subject. The invention involves methods of treating a subject by administering the DNA vectors of the invention, including methods of treating disorders associated with a defect in a target gene.

CROSS REFERENCE

This application is a continuation application of U.S. application Ser.No. 17/675,899, filed Feb. 18, 2022 which is a continuation of U.S.application Ser. No. 17/365,808, filed Jul. 1, 2021, now U.S. Pat. No.11,324,839, issued May 10, 2022 which is continuation of InternationalApplication No. PCT/US2020/051507, filed Sep. 18, 2020 which claimsbenefit to U.S. Provisional Application No. 62/902,084, filed Sep. 18,2019, each of which is entirely incorporated herein by reference for allpurposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Nov. 18, 2022, isnamed 61211-702.305 Sequence Listing.xml and is 57,946 bytes in size.

FIELD OF THE INVENTION

In general, the invention features synthetic DNA vectors.

BACKGROUND

Gene therapy involves transduction of heterologous genes into targetcells to correct a genetic defect underlying a disorder in a subject. Avariety of transduction approaches have been developed for use in genetherapy over the past several decades. For example, traditionalbacterial plasmid DNA vectors represent a versatile tool in genedelivery but can present limitations owing to their bacterial origin.Plasmid DNA vectors include bacterial genes, such as antibioticresistance genes and origins of replication. Additionally, plasmid DNAvectors include bacterial signatures, such as CpG motifs. In addition,the use of bacterial expression systems for producing plasmid DNAvectors involves the risk of introducing contaminating impurities fromthe bacterial host, such as endotoxins or bacterial genomic DNA and RNA,which can lead to loss of gene expression in vivo, e.g., bytranscriptional silencing.

Recombinant adeno-associated viral (rAAV) vectors have an establishedrecord of high-efficiency gene transfer in a variety of model systemsand are now being tested as therapeutic modalities in a wide range ofhuman diseases. Genomes of rAAV vectors can persist in vivo (e.g., inpost-mitotic cells) as circular episomes. After infection,single-stranded rAAV DNA is converted to double stranded circular DNA inthe cell nucleus and persists in an episomal form for the life of thecell. Thus, a substantial benefit of AAV vector systems is the abilityto persist long term in a target cell. On the other hand, AAV vectorscan involve additional drawbacks, such as a limited packaging capacityof about 4.5 Kb, immunogenicity of viral proteins, and manufacturingdifficulties.

Thus, there is a need in the field for versatile and efficient methodsto enhance long-term persistence of gene expression, such as thatprovided by rAAV, while enabling large payloads and reducing the risk ofadverse effects (e.g., inflammation).

SUMMARY

The present invention provides non-viral, circular DNA vectors thatreplicate the in vivo persistence of rAAV vectors. The present DNAvectors are non-immunogenic and are not limited to the AAV packagingcapacity (about 4.5 Kb). The invention also features methods ofproducing the circular DNA vector (e.g., using cell-free methodsproviding improved manufacturing efficiencies over conventionalbacterial-based syntheses), pharmaceutical compositions including thecircular DNA vector, and methods of using the vectors described herein,e.g., for inducing persistent episomal expression of a heterologous geneand for treating a disease associated with a defective gene.

In one aspect, the invention provides an isolated circular DNA vectorincluding one or more heterologous genes encoding a therapeuticreplacement protein, wherein the DNA vector lacks: (a) an origin ofreplication (e.g., a bacterial original of replication) and/or a drugresistance gene; and (b) a recombination site. For example, in someembodiments, the DNA vector lacks an origin of replication, a drugresistance gene, and a recombination site. The therapeutic replacementprotein may be, e.g., an enzyme, a growth factor, a hormone, aninterleukin, an interferon, a cytokine, an anti-apoptosis factor, ananti-diabetic factor, a coagulation factor, an anti-tumor factor, aliver-secreted protein, or a neuroprotective factor. In someembodiments, the enzyme is an epigenetic regulator. In some embodiments,the epigenetic regulator is a histone methyltransferase, a histonedemethylase, a histone acetylase, a DNA methyltransferase, or a DNAdemethylase.

In another aspect, the invention provides an isolated circular DNAvector including one or more heterologous genes encoding anantigen-binding protein. In some embodiments, the DNA vector lacks: (a)an origin of replication (e.g., a bacterial original of replication)and/or a drug resistance gene; and (b) a recombination site. Forexample, in some embodiments, the DNA vector lacks an origin ofreplication, a drug resistance gene, and a recombination site. Theantigen-binding protein may be an antibody or an antigen-bindingfragment thereof. The antigen-binding protein may bind a cytokine, agrowth factor, or a cell-surface protein (e.g., a tumor-associatedantigen). In some embodiments, the antigen-binding protein binds TNF,LT, IFN-α, IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21,EMAP-II, GM-CSF, EGF, HER2, HER3, FGF, PDGF, BDNF, CNTF, CSF, G-CSF,NGF, PEDF, TGF, VEGF, gonadotropin, insulin-like growth factor, CD2,CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80,CD86, CD90, PD-1, PD-L1, amyloid beta, alkaline phosphatase, amyloidprotein A, CCR4, folate receptor, mucin 5AC, PCSK-9,phosphatidyl-serine, or sclerostin. The antigen-binding protein may be amonoclonal antibody, a polyclonal antibody, a multispecific antibody(e.g., a bispecific antibody), and/or an antigen-binding fragment (e.g.,Fab, scFv, scFab, etc.).

In another aspect, the invention provides an isolated circular DNAvector including one or more heterologous genes encoding an enzyme, agrowth factor, a hormone, an interleukin, an interferon, a cytokine, ananti-apoptosis factor, an anti-diabetic factor, a coagulation factor, ananti-tumor factor, a liver-secreted protein, or a neuroprotectivefactor, wherein the DNA vector lacks: (a) an origin of replication(e.g., a bacterial original of replication) and/or a drug resistancegene; and (b) a recombination site. The growth factor may bebrain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor(CNTF), colony stimulating factor (CSF), epidermal growth factor (EGF),fibroblast growth factor (FGF), granulocyte-colony stimulating factor(G-SCF), macrophage-colony stimulating factor (M-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growthfactor (NGF), platelet-derived growth factor (PDGF), pigmentepithelium-derived factor (PEDF), transforming growth factor (TGF; e.g.,TGF-β), vascular endothelial growth factor (VEGF), gonadotropin, or aninsulin-like growth factor. The interleukin (IL) may be IL-1 (e.g.,IL-1β), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, or IL-21. Theinterferon (IFN) may be IFN-α or IFN-γ. The coagulation factor may befactor V, factor VII, factor VIII, factor IX, factor X, factor XI,factor XII, factor XIII, or von Willebrand factor. The neuroprotectivefactor may be selected from the group consisting of a neurotrophin (NT,e.g., NGF, BDNF, NT-3, NT-4, or CNTF), Kifap3, Bcl-xl, Crmp1, Chk.beta.,CALM2, Caly, NPG11, NPT1, Eef1a1, Dhps, Cd151, Morf412, CTGF, LDH-A,Atl1, NPT2, Ehd3, Cox5b, Tuba1a, gamma-actin, Rpsa, NPG3, NPG4, NPG5,NPG6, NPG7, NPG8, NPG9, and NPG10.

In another aspect, the invention provides an isolated circular DNAvector including one or more heterologous genes associated with adisorder selected from the group consisting of an ocular disorder, aliver disorder, a neurological disorder, an immune disorder, a cancer, acardiovascular disorder, a blood coagulation disorder, a lysosomalstorage disorder, or type 2 diabetes, wherein the DNA vector lacks: (a)an origin of replication (e.g., a bacterial original of replication)and/or a drug resistance gene; and (b) a recombination site. Forexample, in some embodiments, the DNA vector lacks an origin ofreplication, a drug resistance gene, and a recombination site.

The disorder may be an ocular disorder that is a retinal dystrophy(e.g., a Mendelian-heritable retinal dystrophy). The retinal dystrophymay be selected from the group consisting of leber's congenitalamaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod conedystrophy, exudative vitreoretinopathy, Joubert Syndrome, congenitalstationary night blindness, type 1C (CSNB-1C), age-related maculardegeneration, retinitis pigmentosa, stickler syndrome, microcephaly andchorioretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, andWagner syndrome. In some embodiments, the disorder is a cancer and theheterologous gene is CD40, CD40L, CD46, XCL1, MDA-7, IL-12, IL-24, orOPCML (opioid binding protein/cell adhesion molecule). The disorder may,in some instances, be a cancer and the heterologous gene is a tumorsuppressor gene. The tumor suppressor gene may be a gene encoding anintracellular protein. The tumor suppressor gene may be a gene encodinga receptor or signal transducer for a secreted hormone or developmentalsignal that inhibits cell proliferation. The tumor suppressor gene alsomay be a gene that encodes a checkpoint control protein. The tumorsuppressor gene may be a gene that encodes a pro-apoptotic protein. Thetumor suppressor gene may be a gene that encodes a DNA repair protein.

In some embodiments, the disorder is a coagulation disorder, such ashemophilia (e.g., hemophilia A or hemophilia B), von Willebrand'sdisease, factor XI deficiency, a fibrinogen disorder, or a vitamin Kdeficiency. The coagulation disorder may be characterized by a mutationin a gene encoding for fibrinogen, prothrombin, factor V, factor VII,factor VIII, factor X, factor XI, factor XIII, or an enzyme involved inposttranslational modifications thereof, or an enzyme involved invitamin K metabolism. In some embodiments, the coagulation disorder ischaracterized by a mutation in a one or more of the following genes:fibrinogen alpha (FGA), fibrinogen beta (FGB), fibrinogen gamma (FGG),factor (F) F2, F5, F7, F10, F11, F13A, F13B, lectin mannose binding 1(LMAN1), multiple coagulation factor deficiency 2 (MCFD2), gammaglutamyl carboxylase (GGCX), or vitamin K epoxide reductase complexsubunit 1 (VKORC1).

The disorder may be a neurological disorder, e.g., a neurodegenerativedisease. The neurodegenerative disease may be selected from the groupconsisting of Alzheimer's disease, Parkinson's disease, or multiplesclerosis. The neurodegenerative disease may be an autoimmune disease ofthe central nervous system (CNS), such as multiple sclerosis,encephalomyelitis, a paraneoplastic syndrome, autoimmune inner eardisease, or opsoclonus myoclonus syndrome. The neurological disorder maybe a cerebral infarction, spinal cord injury, central nervous systemdisorder, a neuropsychiatric disorder, or a channelopathy (e.g.,epilepsy or migraine). The neurological disorder may be an anxietydisorder, a mood disorder, a childhood disorder, a cognitive disorder,schizophrenia, a substance related disorders, or an eating disorders. Insome embodiments, the neurological disorder is a symptom of a cerebralinfarction, stroke, traumatic brain injury, or spinal cord injury.

In some embodiments, the disorder is a lysosomal storage disorder, suchas Tay-Sachs disease, Gaucher disease, Fabry disease, Pompe disease,Niemann-Pick disease, or mucopolysaccharidosis (MPS).

In some embodiments, the disorder is a cardiovascular disorder, such asa degenerative heart disease, a coronary artery disease, an ischemia,angina pectoris, an acute coronary syndrome, a peripheral vasculardisease, a peripheral arterial disease, a cerebrovascular disease, oratherosclerosis. The cardiovascular disorder may be a degenerative heartdisease selected from the group consisting of an ischemiccardiomyopathy, a conduction disease, and a congenital defect.

The disorder may be an immune disorder, e.g., an autoimmune disorder.The autoimmune disorder may be type 1 diabetes, multiple sclerosis,rheumatoid arthritis, lupus, encephalomyelitis, a paraneoplasticsyndrome, autoimmune inner ear disease, or opsoclonus myoclonussyndrome, autoimmune hepatitis, uveitis, autoimmune retinopathy,neuromyelitis optica, psoriatic arthritis, psoriasis, myasthenia gravis,chronic Lyme disease, celiac disease, chronic inflammatory demyelinatingpolyneuropathy, peripheral neuropathy, fibromyalgia, Hashimoto'sthyroiditis, ulcerative colitis, or Kawasaki disease.

The disorder may be a liver disease, e.g., a liver disease selected fromthe group consisting of hepatitis, Alagille syndrome, biliary atresia,liver cancer, cirrhosis, a cystic disease, Caroli's syndrome, congenitalhepatic fibrosis, fatty liver, galactosemia, primary sclerosingcholangitis, tyrosinemia, glycogen storage disease, Wilson's disease,and an endocrine deficiency. The liver disease may be a liver cancerselected from the group consisting of a hepatocellular hyperplasia, ahepatocellular adenomas, a focal nodular hyperplasia, or ahepatocellular carcinoma.

In some embodiments, the disorder is a cancer, such as a blood cancer(e.g., acute lymphoblastic leukemia, acute myeloblastic leukemia,chromic myelogenous leukemia, Hodgkin's disease, multiple myeloma, andnon-Hodgkin's lymphoma) or a solid tissue cancer (e.g., liver cancer,kidney cancer, a breast cancer, a prostate cancer, a gastric cancer, anesophageal cancer, a stomach cancer, an intestinal cancer, a colorectalcancer, a bladder cancer, a head and neck cancer, a skin cancer, or abrain cancer). In some embodiments, the heterologous gene encodes atranscription factor, such as TSHZ2, HOXA2, MEIS2, HOXA3, HAND2, HOXA5,TBX18, PEG3, GLI2, CLOCK, HNF4A, VHL/HIF, WT-1, GSK-3, SPINT2, SMAD2,SMAD3, or SMAD4.

In some embodiments, the disorder is a recessively inherited disorder.In some embodiments, the disorder is a Mendelian-inherited disorder.

In some embodiments of any of the above aspects, the heterologous geneis expressible in a target cell selected from the group consisting of aliver cell, a retinal cell, a stem cell, a neural cell, a muscle cell,or a blood cell. The target cell may be a post-mitotic cell. In someembodiments, the target cell may be a neural cell selected from thegroup consisting of a neuron, an astrocyte, an oligodendrocyte, and aSchwann cell.

In some embodiments of any of the above aspects, the therapeutic proteinis secreted into blood (e.g., when endogenously expressed, the proteinnormally secreted into the blood).

In some embodiments of any of the above aspects, the DNA vector includesa terminal repeat sequence ((e.g., one or more inverted terminal repeat(ITR) sequences (e.g., two ITR sequences) or portion thereof (e.g., twoA elements, B elements, C elements, or D elements), or long terminalrepeat (LTR) sequences (e.g., two LTR sequences)). In some embodiments,the terminal repeat sequence is at least 10 base pairs (bp) in length(e.g., from 10 bp to 500 bp, from 12 bp to 400 bp, from 14 bp to 300 bp,from 16 bp to 250 bp, from 18 bp to 200 bp, from 20 bp to 180 bp, from25 bp to 170 bp, from 30 bp to 160 bp, or from 50 bp to 150 bp, e.g.,from 10 bp to 15 bp, from 15 bp to 20 bp, from 20 bp to 25 bp, from 25bp to 30 bp, from 30 bp to 35 bp, from 35 bp to 40 bp, from 40 bp to 45bp, from 45 bp to 50 bp, from 50 bp to 55 bp, from 55 bp to 60 bp, from60 bp to 65 bp, from 65 bp to 70 bp, from 70 bp to 80 bp, from 80 bp to90 bp, from 90 bp to 100 bp, from 100 bp to 150 bp, from 150 bp to 200bp, from 200 bp to 300 bp, from 300 bp to 400 bp, or from 400 bp to 500bp, e.g., 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp,19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29bp, 30 bp, 31 bp, 32 bp, 33 bp, 34 bp, 35 bp, 36 bp, 37 bp, 38 bp, 39bp, 40 bp, 41 bp, 42 bp, 43 bp, 44 bp, 45 bp, 46 bp, 47 bp, 48 bp, 49bp, 50 bp, 51 bp, 52 bp, 53 bp, 54 bp, 55 bp, 56 bp, 57 bp, 58 bp, 59bp, 60 bp, 61 bp, 62 bp, 63 bp, 64 bp, 65 bp, 66 bp, 67 bp, 68 bp, 69bp, 70 bp, 71 bp, 72 bp, 73 bp, 74 bp, 75 bp, 76 bp, 77 bp, 78 bp, 79bp, 80 bp, 81 bp, 82 bp, 83 bp, 84 bp, 85 bp, 86 bp, 87 bp, 88 bp, 89bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99bp, 100 bp, 101 bp, 102 bp, 103 bp, 104 bp, 105 bp, 106 bp, 107 bp, 108bp, 109 bp, 110 bp, 111 bp, 112 bp, 113 bp, 114 bp, 115 bp, 116 bp, 117bp, 118 bp, 119 bp, 120 bp, 121 bp, 122 bp, 123 bp, 124 bp, 125 bp, 126bp, 127 bp, 128 bp, 129 bp, 130 bp, 131 bp, 132 bp, 133 bp, 134 bp, 135bp, 136 bp, 137 bp, 138 bp, 139 bp, 140 bp, 141 bp, 142 bp, 143 bp, 144bp, 145 bp, 146 bp, 147 bp, 148 bp, 149 bp, 150 bp, or more). In someembodiments, the DNA vector includes a DD element.

In some embodiments of any of the above aspects, the DNA vector includesa promoter sequence upstream of the one or more heterologous genes.

In some embodiments of any of the above aspects, the DNA vector includesa polyadenylation site downstream of the one or more heterologous genes.

In some embodiments of any of the above aspects, the one or moreheterologous genes includes a trans-splicing molecule or a portionthereof (e.g., a binding domain).

In another aspect, the invention provides an isolated circular DNAvector having one or more therapeutic nucleic acids. Such an isolatedcircular DNA vector lacks an origin of replication and/or a drugresistance gene; lacks a recombination site; and comprises a terminalrepeat sequence (e.g., a DD element). In some embodiments, thetherapeutic nucleic acid is an siRNA, an shRNA, an miRNA, or a CRISPRimolecule. In some embodiments, the terminal repeat sequence (e.g., DDelement) is at least 10 bp in length.

In some embodiments of any of the above aspects, the vector includes asuicide gene. In some embodiments of any of the above aspects, the DNAvector lacks bacterial plasmid DNA. In some embodiments, the DNA vectorincludes one or more unmethylated GATC sequences, one or moreunmethylated CCAGG sequences, and/or one or more CCTGG sequences.Additionally or alternatively, the DNA vector may (a) lacks animmunogenic bacterial signature; (b) lack an RNA polymerase arrest site;and/or (c) be substantially devoid of CpG islands.

In some embodiments of any of the above aspects, the heterologous geneis greater than 4.5 Kb in length (e.g., the one or more heterologousgenes, together or each alone, are from 4.5 Kb to 25 Kb, from 4.6 Kb to24 Kb, from 4.7 Kb to 23 Kb, from 4.8 Kb to 22 Kb, from 4.9 Kb to 21 Kb,from 5.0 Kb to 20 Kb, from 5.5 Kb to 18 Kb, from 6.0 Kb to 17 Kb, from6.5 Kb to 16 Kb, from 7.0 Kb to 15 Kb, from 7.5 Kb to 14 Kb, from 8.0 Kbto 13 Kb, from 8.5 Kb to 12.5 Kb, from 9.0 Kb to 12.0 Kb, from 9.5 Kb to11.5 Kb, or from 10.0 Kb to 11.0 Kb in length, e.g., from 4.5 Kb to 8Kb, from 8 Kb to 10 Kb, from 10 Kb to 15 Kb, from 15 Kb to 20 Kb inlength, or greater, e.g., from 4.5 Kb to 5.0 Kb, from 5.0 Kb to 5.5 Kb,from 5.5 Kb to 6.0 Kb, from 6.0 Kb to 6.5 Kb, from 6.5 Kb to 7.0 Kb,from 7.0 Kb to 7.5 Kb, from 7.5 Kb to 8.0 Kb, from 8.0 Kb to 8.5 Kb,from 8.5 Kb to 9.0 Kb, from 9.0 Kb to 9.5 Kb, from 9.5 Kb to 10 Kb, from10 Kb to 10.5 Kb, from 10.5 Kb to 11 Kb, from 11 Kb to 11.5 Kb, from11.5 Kb to 12 Kb, from 12 Kb to 12.5 Kb, from 12.5 Kb to 13 Kb, from 13Kb to 13.5 Kb, from 13.5 Kb to 14 Kb, from 14 Kb to 14.5 Kb, from 14.5Kb to 15 Kb, from 15 Kb to 15.5 Kb, from 15.5 Kb to 16 Kb, from 16 Kb to16.5 Kb, from 16.5 Kb to 17 Kb, from 17 Kb to 17.5 Kb, from 17.5 Kb to18 Kb, from 18 Kb to 18.5 Kb, from 18.5 Kb to 19 Kb, from 19 Kb to 19.5Kb, from 19.5 Kb to 20 Kb, from 20 Kb to 21 Kb, from 21 Kb to 22 Kb,from 22 Kb to 23 Kb, from 23 Kb to 24 Kb, from 24 Kb to 25 Kb in length,or greater, e.g., about 4.5 Kb, about 5.0 Kb, about 5.5 Kb, about 6.0Kb, about 6.5 Kb, about 7.0 Kb, about 7.5 Kb, about 8.0 Kb, about 8.5Kb, about 9.0 Kb, about 9.5 Kb, about 10 Kb, about 11 Kb, about 12 Kb,about 13 Kb, about 14 Kb, about 15 Kb, about 16 Kb, about 17 Kb, about18 Kb, about 19 Kb, about 20 Kb in length, or greater).

In some embodiments of any of the above aspects, the DNA vector isdouble stranded. Additionally or alternatively, the double strandedvector may be monomeric and/or supercoiled.

In some embodiments, the DNA vector is covalently closed.

In another aspect, the invention provides a composition (e.g., apharmaceutical composition) comprising a plurality of the DNA vectors ofany of the preceding aspects. In some embodiments, at least 50% of theplurality of the DNA vectors of the composition (e.g., pharmaceuticalcomposition) includes one or more unmethylated GATC sequences, one ormore unmethylated CCAGG sequences, and/or one or more CCTGG sequences.

In another aspect, the invention provides an isolated linear DNAmolecule including a plurality of identical amplicons (e.g., aconcatamer), wherein each of the plurality of identical ampliconsincludes a heterologous gene encoding a therapeutic replacement protein.The DNA molecule lacks: (a) an origin of replication (e.g., a bacterialoriginal of replication) and/or a drug resistance gene; and (b) arecombination site. For example, in some embodiments, the isolatedlinear DNA molecule lacks an origin of replication, a drug resistancegene, and a recombination site. In some embodiments, the isolated linearDNA molecule includes restriction enzyme sites, e.g., wherein eachrestriction enzyme site is positioned between the heterologous gene anda terminal repeat sequence. The therapeutic replacement protein may be,e.g., an enzyme, a growth factor, a hormone, an interleukin, aninterferon, a cytokine, an anti-apoptosis factor, an anti-diabeticfactor, a coagulation factor, an anti-tumor factor, a liver-secretedprotein, or a neuroprotective factor.

In another aspect, the invention provides an isolated linear DNAmolecule including a plurality of identical amplicons, wherein each ofthe plurality of identical amplicons includes one or more heterologousgenes encoding an antigen-binding protein, wherein the DNA vector lacks:(a) an origin of replication (e.g., a bacterial original of replication)and/or a drug resistance gene; and (b) a recombination site. Forexample, in some embodiments, the isolated linear DNA molecule lacks anorigin of replication, a drug resistance gene, and a recombination site.In some embodiments, the isolated linear DNA molecule includesrestriction enzyme sites, e.g., wherein each restriction enzyme site ispositioned between the heterologous gene and a terminal repeat sequence.

In some embodiments, the antigen-binding protein is an antibody or anantigen-binding fragment thereof. The antigen-binding protein may bind acytokine, a growth factor, or a cell-surface protein (e.g., atumor-associated antigen). In some embodiments, the antigen-bindingprotein binds tumor necrosis factor (TNF), large T antigen (LT), IFN-α,IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21,endothelial-monocyte-activating polypeptide II (EMAP-II), GM-CSF, EGF,human epidermal growth factor 2 (HER2), HER3, FGF, PDGF, BDNF, CNTF,CSF, G-CSF, NGF, PEDF, TGF, VEGF, gonadotropin, insulin-like growthfactor, CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45,CD69, CD80, CD86, CD90, programmed death-1 (PD-1), programmeddeath-ligand 1 (PD-L1), amyloid beta, alkaline phosphatase, amyloidprotein A, CC chemokine receptor 4 (CCR4), folate receptor, mucin 5AC,proprotein convertase subtilisin/kexin type 9 (PCSK-9),phosphatidyl-serine, or sclerostin. The antigen-binding protein may be amonoclonal antibody, a polyclonal antibody, a multispecific antibody(e.g., a bispecific antibody), and/or an antigen-binding fragment (e.g.,Fab, scFv, scFab, etc.).

In another aspect, the invention provides an isolated linear DNAmolecule including a plurality of identical amplicons, wherein each ofthe plurality of identical amplicons includes one or more heterologousgenes encoding an enzyme, a growth factor, a hormone, an interleukin, aninterferon, a cytokine, an anti-apoptosis factor, an anti-diabeticfactor, a coagulation factor, an anti-tumor factor, a liver-secretedprotein, or a neuroprotective factor, wherein the DNA molecule lacks:(a) an origin of replication (e.g., a bacterial original of replication)and/or a drug resistance gene; and (b) a recombination site. Forexample, in some embodiments, the isolated linear DNA molecule lacks anorigin of replication, a drug resistance gene, and a recombination site.In some embodiments, the isolated linear DNA molecule includesrestriction enzyme sites, e.g., wherein each restriction enzyme site ispositioned between the heterologous gene and a terminal repeat sequence.

The growth factor may be BDNF, CNTF, CSF, EGF, FGF, G-SCF, M-CSF,GM-CSF, NGF, PDGF, PEDF, TGF, VEGF, gonadotropin, or an insulin-likegrowth factor. The interleukin may be is IL-1 (e.g., IL-1β), IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, IL-16, IL-17, IL-18, or IL-21. The interferon may be IFN-α orIFN-γ. The coagulation factor may be factor V, factor VII, factor VIII,factor IX, factor X, factor XI, factor XII, factor XIII, or vonWillebrand factor. The neuroprotective factor may be selected from thegroup consisting of a neurotrophin, Kifap3, Bcl-xl, Crmp1, Chk.beta.,CALM2, Caly, NPG11, NPT1, Eef1a1, Dhps, Cd151, Morf412, CTGF, LDH-A,Atl1, NPT2, Ehd3, Cox5b, Tuba1a, gamma-actin, Rpsa, NPG3, NPG4, NPG5,NPG6, NPG7, NPG8, NPG9, and NPG10. The neurotrophin may be selected fromthe group consisting of NGF, BDNF, NT-3, NT-4, and CNTF.

In another aspect, the invention provides an isolated linear DNAmolecule including a plurality of identical amplicons, wherein each ofthe plurality of identical amplicons includes one or more heterologousgenes associated with a disorder selected from the group consisting ofan ocular disorder, a liver disorder, a neurological disorder, an immunedisorder, a cancer, a cardiovascular disorder, a blood coagulationdisorder, a lysosomal storage disorder, or type 2 diabetes, wherein theDNA molecule lacks: (a) an origin of replication (e.g., a bacterialoriginal of replication) and/or a drug resistance gene; and (b) arecombination site. For example, in some embodiments, the isolatedlinear DNA molecule lacks an origin of replication, a drug resistancegene, and a recombination site. In some embodiments, the isolated linearDNA molecule includes restriction enzyme sites, e.g., wherein eachrestriction enzyme site is positioned between the heterologous gene anda terminal repeat sequence.

The disorder may be an ocular disorder that is a retinal dystrophy(e.g., a Mendelian-heritable retinal dystrophy). The retinal dystrophymay be selected from the group consisting of leber's congenitalamaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod conedystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C,age-related macular degeneration, retinitis pigmentosa, sticklersyndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB2, Usher syndrome, and Wagner syndrome.

In some embodiments, the disorder is a coagulation disorder, such ashemophilia (e.g., hemophilia A or hemophilia B), von Willebrand'sdisease, factor XI deficiency, a fibrinogen disorder, or a vitamin Kdeficiency. The coagulation disorder may be characterized by a mutationin a gene encoding for fibrinogen, prothrombin, factor V, factor VII,factor VIII, factor X, factor XI, factor XIII, or an enzyme involved inposttranslational modifications thereof, or an enzyme involved invitamin K metabolism. In some embodiments, the coagulation disorder ischaracterized by a mutation in FGA, FGB, FGG, F2, F5, F7, F10, F11,F13A, F13B, LMAN1, MCFD2, GGCX, or VKORC1.

The disorder may be a neurological disorder, e.g., a neurodegenerativedisease. The neurodegenerative disease may be selected from the groupconsisting of Alzheimer's disease, Parkinson's disease, or multiplesclerosis. The neurodegenerative disease may be an autoimmune disease ofthe central nervous system (CNS), such as multiple sclerosis,encephalomyelitis, a paraneoplastic syndrome, autoimmune inner eardisease, or opsoclonus myoclonus syndrome. The neurological disorder maybe a cerebral infarction, spinal cord injury, central nervous systemdisorder, a neuropsychiatric disorder, or a channelopathy (e.g.,epilepsy or migraine). The neurological disorder may be an anxietydisorder, a mood disorder, a childhood disorder, a cognitive disorder,schizophrenia, a substance related disorders, or an eating disorders. Insome embodiments, the neurological disorder is a symptom of a cerebralinfarction, stroke, traumatic brain injury, or spinal cord injury.

In some embodiments, the disorder is a lysosomal storage disorder, suchas Tay-Sachs disease, Gaucher disease, Fabry disease, Pompe disease,Niemann-Pick disease, or mucopolysaccharidosis (MPS).

In some embodiments, the disorder is a cardiovascular disorder, such asa degenerative heart disease, a coronary artery disease, an ischemia,angina pectoris, an acute coronary syndrome, a peripheral vasculardisease, a peripheral arterial disease, a cerebrovascular disease, oratherosclerosis. The cardiovascular disorder may be a degenerative heartdisease selected from the group consisting of an ischemiccardiomyopathy, a conduction disease, and a congenital defect.

The disorder may be an immune disorder, e.g., an autoimmune disorder.The autoimmune disorder may be type 1 diabetes, multiple sclerosis,rheumatoid arthritis, lupus, encephalomyelitis, a paraneoplasticsyndrome, autoimmune inner ear disease, or opsoclonus myoclonussyndrome, autoimmune hepatitis, uveitis, autoimmune retinopathy,neuromyelitis optica, psoriatic arthritis, psoriasis, myasthenia gravis,chronic Lyme disease, celiac disease, chronic inflammatory demyelinatingpolyneuropathy, peripheral neuropathy, fibromyalgia, Hashimoto'sthyroiditis, ulcerative colitis, or Kawasaki disease.

The disorder may be a liver disease, e.g., a liver disease selected fromthe group consisting of hepatitis, Alagille syndrome, biliary atresia,liver cancer, cirrhosis, a cystic disease, Caroli's syndrome, congenitalhepatic fibrosis, fatty liver, galactosemia, primary sclerosingcholangitis, tyrosinemia, glycogen storage disease, Wilson's disease,and an endocrine deficiency. The liver disease may be a liver cancerselected from the group consisting of a hepatocellular hyperplasia, ahepatocellular adenomas, a focal nodular hyperplasia, or ahepatocellular carcinoma.

In some embodiments, the disorder is a cancer, such as a blood cancer(e.g., acute lymphoblastic leukemia, acute myeloblastic leukemia,chromic myelogenous leukemia, Hodgkin's disease, multiple myeloma, andnon-Hodgkin's lymphoma) or a solid tissue cancer (e.g., liver cancer,kidney cancer, a breast cancer, a prostate cancer, a gastric cancer, anesophageal cancer, a stomach cancer, an intestinal cancer, a colorectalcancer, a bladder cancer, a head and neck cancer, a skin cancer, or abrain cancer).

In some embodiments, the disorder is a recessively inherited disorder.In some embodiments, the disorder is a Mendelian-inherited disorder.

In another aspect, the invention provides an isolated linear DNAmolecule having a plurality of identical amplicons, wherein each of theplurality of identical amplicons includes one or more therapeuticnucleic acids. In some embodiments, the DNA molecule (a) lacks an originof replication and/or a drug resistance gene; (b) lacks a recombinationsite; and (c) comprises a terminal repeat sequence. For example, in someembodiments, the isolated linear DNA molecule lacks an origin ofreplication, a drug resistance gene, and a recombination site. In someembodiments, the isolated linear DNA molecule includes restrictionenzyme sites, e.g., wherein each restriction enzyme site is positionedbetween the heterologous gene and a terminal repeat sequence.

In some embodiments, the isolated linear DNA molecule includes aterminal repeat sequence (e.g., one or more inverted terminal repeat(ITR) sequences (e.g., two ITR sequences) or portion thereof (e.g., twoA elements, B elements, C elements, or D elements), or long terminalrepeat (LTR) sequences (e.g., two LTR sequences)). In some embodiments,the terminal repeat sequence is at least 10 base pairs (bp) in length(e.g., from 10 bp to 500 bp, from 12 bp to 400 bp, from 14 bp to 300 bp,from 16 bp to 250 bp, from 18 bp to 200 bp, from 20 bp to 180 bp, from25 bp to 170 bp, from 30 bp to 160 bp, or from 50 bp to 150 bp, e.g.,from 10 bp to 15 bp, from 15 bp to 20 bp, from 20 bp to 25 bp, from 25bp to 30 bp, from 30 bp to 35 bp, from 35 bp to 40 bp, from 40 bp to 45bp, from 45 bp to 50 bp, from 50 bp to 55 bp, from 55 bp to 60 bp, from60 bp to 65 bp, from 65 bp to 70 bp, from 70 bp to 80 bp, from 80 bp to90 bp, from 90 bp to 100 bp, from 100 bp to 150 bp, from 150 bp to 200bp, from 200 bp to 300 bp, from 300 bp to 400 bp, or from 400 bp to 500bp, e.g., 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp,19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29bp, 30 bp, 31 bp, 32 bp, 33 bp, 34 bp, 35 bp, 36 bp, 37 bp, 38 bp, 39bp, 40 bp, 41 bp, 42 bp, 43 bp, 44 bp, 45 bp, 46 bp, 47 bp, 48 bp, 49bp, 50 bp, 51 bp, 52 bp, 53 bp, 54 bp, 55 bp, 56 bp, 57 bp, 58 bp, 59bp, 60 bp, 61 bp, 62 bp, 63 bp, 64 bp, 65 bp, 66 bp, 67 bp, 68 bp, 69bp, 70 bp, 71 bp, 72 bp, 73 bp, 74 bp, 75 bp, 76 bp, 77 bp, 78 bp, 79bp, 80 bp, 81 bp, 82 bp, 83 bp, 84 bp, 85 bp, 86 bp, 87 bp, 88 bp, 89bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99bp, 100 bp, 101 bp, 102 bp, 103 bp, 104 bp, 105 bp, 106 bp, 107 bp, 108bp, 109 bp, 110 bp, 111 bp, 112 bp, 113 bp, 114 bp, 115 bp, 116 bp, 117bp, 118 bp, 119 bp, 120 bp, 121 bp, 122 bp, 123 bp, 124 bp, 125 bp, 126bp, 127 bp, 128 bp, 129 bp, 130 bp, 131 bp, 132 bp, 133 bp, 134 bp, 135bp, 136 bp, 137 bp, 138 bp, 139 bp, 140 bp, 141 bp, 142 bp, 143 bp, 144bp, 145 bp, 146 bp, 147 bp, 148 bp, 149 bp, 150 bp, or more). In someembodiments, isolated linear DNA molecule includes a DD element. In someembodiments, the isolated linear DNA molecule includes a promotersequence upstream of the one or more heterologous genes. In someembodiments, the isolated DNA molecule includes a polyadenylation sitedownstream of the one or more heterologous genes. In some embodiments,the one or more heterologous genes includes a trans-splicing molecule ora portion thereof (e.g., a binding domain).

In another aspect, the invention provides a method of producing theisolated DNA vector of any of the embodiments by: (i) providing a sampleincluding a circular DNA vector including an AAV genome, wherein the AAVgenome includes the heterologous gene; (ii) amplifying the AAV genomeusing polymerase-mediated rolling-circle amplification to generate alinear concatamer; (iii) digesting the concatamer using a restrictionenzyme to generate multiple AAV genomes; and (iv) allowing each of themultiple AAV genomes to self-ligate to produce an isolated DNA vectorincluding the heterologous gene.

In another aspect, the invention provides a method of producing theisolated DNA vector of any one of the above embodiments by: (i)providing a sample including a circular DNA vector including an AAVgenome, wherein the AAV genome includes the heterologous gene and aterminal repeat sequence; (ii) amplifying the AAV genome using a firstpolymerase-mediated rolling-circle amplification to generate a firstlinear concatamer; (iii) digesting the first linear concatamer using arestriction enzyme to generate a first AAV genome; (iv) cloning thefirst AAV genome into a plasmid vector; (v) identifying a plasmid cloneincluding a terminal repeat sequence; (vi) digesting the plasmid cloneincluding the terminal repeat sequence to generate a second AAV genome;(vii) allowing the second AAV genome to self-ligate to produce acircular DNA template; (viii) amplifying the circular DNA template usingsecond polymerase-mediated rolling-circle amplification to generate asecond linear concatamer; (ix) digesting the second linear concatamerusing a restriction enzyme to generate a third AAV genome; and (x)allowing the third AAV genome to self-ligate to produce an isolated DNAvector including the heterologous gene and the terminal repeat sequence.

In another aspect, the invention provides a cell-free method ofproducing the isolated DNA vector of any of the above embodiments by:(i) providing a sample including a circular DNA vector including an AAVgenome, wherein the AAV genome includes the heterologous gene; (ii)amplifying the AAV genome using polymerase-mediated rolling-circleamplification to generate a linear concatamer; (iii) digesting theconcatamer using a restriction enzyme to generate an AAV genome; and(iv) allowing the AAV genome to self-ligate to produce the isolated DNAvector including the heterologous gene. In some embodiments, the methodfurther includes column purifying the isolated DNA vector including theheterologous gene to purify supercoiled DNA from the isolated DNAvector.

In another aspect, the invention provides a method of producing theisolated DNA vector of any of the above embodiments by: (i) providing asample including a circular DNA vector including an AAV genome, whereinthe AAV genome includes the heterologous gene and a DD element; (ii)amplifying the AAV genome using polymerase-mediated rolling-circleamplification to generate a linear concatamer; (iii) digesting theconcatamer using a restriction enzyme to generate multiple AAV genomes;and (iv) allowing each of the multiple AAV genomes to self-ligate toproduce an isolated DNA vector including the heterologous gene and theDD element.

In another aspect, the invention provides a method of producing theisolated DNA vector of any one of the above embodiments by: (i)providing a sample including a circular DNA vector including an AAVgenome, wherein the AAV genome includes the heterologous gene and a DDelement; (ii) amplifying the AAV genome using a firstpolymerase-mediated rolling-circle amplification to generate a firstlinear concatamer; (iii) digesting the first linear concatamer using arestriction enzyme to generate a first AAV genome; (iv) cloning thefirst AAV genome into a plasmid vector; (v) identifying a plasmid cloneincluding a DD element; (vi) digesting the plasmid clone including theDD element to generate a second AAV genome; (vii) allowing the secondAAV genome to self-ligate to produce a circular DNA template; (viii)amplifying the circular DNA template using second polymerase-mediatedrolling-circle amplification to generate a second linear concatamer;(ix) digesting the second linear concatamer using a restriction enzymeto generate a third AAV genome; and (x) allowing the third AAV genome toself-ligate to produce an isolated DNA vector including the heterologousgene and the DD element.

In another aspect, the invention provides a cell-free method ofproducing the isolated DNA vector of the above embodiments by: (i)providing a sample including a circular DNA vector including an AAVgenome, wherein the AAV genome includes the heterologous gene and a DDelement; (ii) amplifying the AAV genome using polymerase-mediatedrolling-circle amplification to generate a linear concatamer; (iii)digesting the concatamer using a restriction enzyme to generate an AAVgenome; and (iv) allowing the AAV genome to self-ligate to produce atherapeutic DNA vector including the heterologous gene and the DDelement.

In some embodiments of any of the aforementioned methods of producingthe isolated DNA vector, the AAV genome includes a terminal repeatsequence (e.g., one or more inverted terminal repeat (ITR) sequences(e.g., two ITR sequences) or portion thereof (e.g., two A elements, Belements, C elements, or D elements), or long terminal repeat (LTR)sequences (e.g., two LTR sequences)). In some embodiments, the terminalrepeat sequence is at least 10 base pairs (bp) in length (e.g., from 10bp to 500 bp, from 12 bp to 400 bp, from 14 bp to 300 bp, from 16 bp to250 bp, from 18 bp to 200 bp, from 20 bp to 180 bp, from 25 bp to 170bp, from 30 bp to 160 bp, or from 50 bp to 150 bp, e.g., from 10 bp to15 bp, from 15 bp to 20 bp, from 20 bp to 25 bp, from 25 bp to 30 bp,from 30 bp to 35 bp, from 35 bp to 40 bp, from 40 bp to 45 bp, from 45bp to 50 bp, from 50 bp to 55 bp, from 55 bp to 60 bp, from 60 bp to 65bp, from 65 bp to 70 bp, from 70 bp to 80 bp, from 80 bp to 90 bp, from90 bp to 100 bp, from 100 bp to 150 bp, from 150 bp to 200 bp, from 200bp to 300 bp, from 300 bp to 400 bp, or from 400 bp to 500 bp, e.g., 10bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29 bp, 30bp, 31 bp, 32 bp, 33 bp, 34 bp, 35 bp, 36 bp, 37 bp, 38 bp, 39 bp, 40bp, 41 bp, 42 bp, 43 bp, 44 bp, 45 bp, 46 bp, 47 bp, 48 bp, 49 bp, 50bp, 51 bp, 52 bp, 53 bp, 54 bp, 55 bp, 56 bp, 57 bp, 58 bp, 59 bp, 60bp, 61 bp, 62 bp, 63 bp, 64 bp, 65 bp, 66 bp, 67 bp, 68 bp, 69 bp, 70bp, 71 bp, 72 bp, 73 bp, 74 bp, 75 bp, 76 bp, 77 bp, 78 bp, 79 bp, 80bp, 81 bp, 82 bp, 83 bp, 84 bp, 85 bp, 86 bp, 87 bp, 88 bp, 89 bp, 90bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, 100bp, 101 bp, 102 bp, 103 bp, 104 bp, 105 bp, 106 bp, 107 bp, 108 bp, 109bp, 110 bp, 111 bp, 112 bp, 113 bp, 114 bp, 115 bp, 116 bp, 117 bp, 118bp, 119 bp, 120 bp, 121 bp, 122 bp, 123 bp, 124 bp, 125 bp, 126 bp, 127bp, 128 bp, 129 bp, 130 bp, 131 bp, 132 bp, 133 bp, 134 bp, 135 bp, 136bp, 137 bp, 138 bp, 139 bp, 140 bp, 141 bp, 142 bp, 143 bp, 144 bp, 145bp, 146 bp, 147 bp, 148 bp, 149 bp, 150 bp, or more). In someembodiments, the terminal repeat sequence includes a DD element. In someembodiments, the method further includes column purifying the isolatedDNA vector including the heterologous gene to purify supercoiled DNAfrom the isolated DNA vector.

In some embodiments of any of the above methods, the polymerase-mediatedrolling-circle amplification is isothermal rolling-circle amplification.The polymerase may be Phi29 DNA polymerase.

In another aspect, the invention features a pharmaceutical compositionincluding the DNA vector of any of the above embodiments and apharmaceutically acceptable carrier. The pharmaceutical composition maybe non-immunogenic.

In another aspect, the invention features a method of inducing episomalexpression (e.g., persistent episomal expression) of a heterologous genein a subject in need thereof by administering to the subject theisolated DNA vector or composition (e.g., pharmaceutical composition) ofany of the above embodiments.

In another aspect, the invention features a method of treating adisorder in a subject by administering to the subject the isolated DNAvector or composition (e.g., pharmaceutical composition) of any of theabove embodiments in a therapeutically effective amount.

The isolated DNA vector or composition thereof may be administeredrepeatedly. The isolated DNA vector or the composition may beadministered systemically. The isolated DNA vector or the compositionmay be administered intravenously. The isolated DNA vector or thepharmaceutical composition may be administered locally. The isolated DNAvector or the pharmaceutical composition may be administeredintravitreally. In some embodiments, the disorder is an ocular disorder.The ocular disorder may be a Mendelian-heritable retinal dystrophy. Theocular disorder may be LCA, Stargardt Disease, pseudoxanthoma elasticum,rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome,CSNB-1C, age-related macular degeneration, retinitis pigmentosa,stickler syndrome, microcephaly and chorioretinopathy, retinitispigmentosa, CSNB 2, Usher syndrome, or Wagner syndrome.

In some embodiments, the isolated DNA vector or composition thereof isadministered systemically. In some embodiments, the disorder is acoagulation disorder (e.g., hemophilia (e.g., hemophilia A or hemophiliaB), von Willebrand's disease, factor XI deficiency, a fibrinogendisorder, or a vitamin K deficiency).

In another aspect, the invention provides an isolated circular DNAvector including one or more heterologous genes, wherein the DNA vectorlacks an origin of replication (e.g., a bacterial origin of replication)and/or a drug-resistance gene (e.g., as part of a bacterial plasmid).For example, an isolated circular DNA vector including one or moreheterologous genes may lack an origin of replication (e.g., a bacterialorigin of replication). Additionally, or alternatively, an isolatedcircular DNA vector including one or more heterologous genes may lack adrug-resistance gene (e.g., as part of a bacterial plasmid). In someembodiments, an isolated circular DNA vector including one or moreheterologous genes may lack an origin of replication (e.g., a bacterialorigin of replication) and a drug-resistance gene (e.g., as part of abacterial plasmid). In some embodiments, the DNA molecule lacksbacterial plasmid DNA. In some embodiments, the DNA vector lacks animmunogenic bacterial signature (e.g., one or more bacterial-associatedCpG motifs, e.g., unmethylated CpG motifs, e.g., CpG islands). In someembodiments, the DNA vector lacks an RNA polymerase arrest site (e.g.,an RNA polymerase II (RNAPII) arrest site).

In some embodiments, the isolated circular DNA vector includes one ormore heterologous genes encoding a therapeutic protein configured totreat a Mendelian-heritable retinal dystrophy (e.g., Leber's congenitalamaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod conedystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C,retinitis pigmentosa, stickler syndrome, microcephaly andchorioretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, andWagner syndrome). For example, the one or more heterologous genes can beABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, COL11A1,TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1.

In another aspect, the invention provides an isolated circular DNAvector having one or more heterologous genes selected from the groupconsisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172,COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1,wherein the DNA vector lacks an origin of replication and/or a drugresistance gene. In some embodiments, the one or more heterologous genesencode a therapeutic protein configured to treat a retinal dystrophy(e.g., a Mendelian-heritable retinal dystrophy, e.g., a retinaldystrophy selected from the group consisting of LCA, Stargardt Disease,pseudoxanthoma elasticum, rod cone dystrophy, exudativevitreoretinopathy, Joubert Syndrome, CSNB-1C, retinitis pigmentosa,stickler syndrome, microcephaly and chorioretinopathy, retinitispigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome).

In another aspect, provided herein is an isolated circular DNA vectorhaving one or more heterologous genes encoding a therapeutic protein(e.g., an antibody or portion thereof, a growth factor, an interleukin,an interferon, an anti-apoptosis factor, a cytokine, or an anti-diabeticfactor), wherein the DNA vector lacks an origin of replication and/or adrug resistance gene.

In another aspect, the invention provides an isolated circular DNAvector having one or more heterologous genes including a trans-splicingmolecule or a portion thereof (e.g., a binding domain), wherein the DNAvector lacks an origin of replication and/or a drug resistance gene.

In another aspect, the invention provides an isolated circular DNAvector comprising one or more heterologous genes encoding aliver-secreted therapeutic protein, wherein the DNA vector lacks anorigin of replication and/or a drug resistance gene. In someembodiments, the therapeutic protein is secreted into blood.

In another aspect, the invention provides an isolated circular DNAvector comprising one or more heterologous genes, wherein the DNAvector: (a) includes a terminal repeat sequence; and (b) lacks an originof replication and/or a drug resistance gene.

In yet another aspect, the invention provides an isolated linear DNAmolecule having a plurality of identical amplicons, wherein each of theplurality of identical amplicons comprises a heterologous gene encodinga therapeutic protein (e.g., a therapeutic protein configured to treat aretinal dystrophy, e.g., a Mendelian-heritable retinal dystrophy),wherein the DNA molecule lacks: (a) an origin of replication and/or adrug resistance gene; and (b) a recombination site. In some embodiments,the retinal dystrophy is selected from the group consisting of LCA,Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy,exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C, retinitispigmentosa, age related macular degeneration (AMD), stickler syndrome,microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB 2, Ushersyndrome, and Wagner syndrome. In some embodiments, the one or moreheterologous genes are selected from the group consisting of ABCA4,CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172, C3, COL11A1,TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1.

In another aspect, the invention provides an isolated linear DNAmolecule having a plurality of identical amplicons, wherein each of theplurality of identical amplicons including a heterologous gene selectedfrom the group consisting of ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A,TRPM1, IFT-172, C3, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN,USH2A, and HMCN1, wherein the DNA molecule lacks: (a) an origin ofreplication and/or a drug resistance gene; and (b) a recombination site.In some embodiments, the heterologous gene encodes a therapeutic proteinconfigured to treat a retinal dystrophy (e.g., a Mendelian-heritableretinal dystrophy, e.g., LCA, Stargardt Disease, pseudoxanthomaelasticum, rod cone dystrophy, exudative vitreoretinopathy, JoubertSyndrome, CSNB-1C, retinitis pigmentosa, AMD, stickler syndrome,microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB 2, Ushersyndrome, or Wagner syndrome).

In another aspect, provided herein is an isolated linear DNA moleculehaving a plurality of identical amplicons, wherein each of the pluralityof identical amplicons includes a heterologous gene encoding antibody orportion thereof, a coagulation factor, an enzyme, a growth factor, ahormone, an interleukin, an interferon, an anti-apoptosis factor, ananti-tumor factor, a cytokine, and an anti-diabetic factor, wherein theDNA molecule lacks: (a) an origin of replication and/or a drugresistance gene; and (b) a recombination site.

In yet another aspect, the invention features an isolated linear DNAmolecule having a plurality of identical amplicons, wherein each of theplurality of identical amplicons includes a heterologous gene comprisinga trans-splicing molecule or a portion thereof (e.g., a binding domain),wherein the DNA molecule lacks: (a) an origin of replication and/or adrug resistance gene; and (b) a recombination site.

In another aspect, the invention provides an isolated linear DNAmolecule having a plurality of identical amplicons, wherein each of theplurality of identical amplicons includes a heterologous gene encoding aliver-secreted therapeutic protein (e.g., a therapeutic protein secretedinto blood), wherein the DNA molecule lacks an origin of replicationand/or a drug resistance gene.

In some embodiments of any of the preceding aspects, the circular DNAvector or linear DNA molecule further includes one or more terminalrepeat sequences (e.g., one or more inverted terminal repeat (ITR)sequences (e.g., two ITR sequences) or portion thereof (e.g., two Aelements, B elements, C elements, or D elements), or long terminalrepeat (LTR) sequences (e.g., two LTR sequences)). In some embodiments,the terminal repeat sequence is at least 10 base pairs (bp) in length(e.g., from 10 bp to 500 bp, from 12 bp to 400 bp, from 14 bp to 300 bp,from 16 bp to 250 bp, from 18 bp to 200 bp, from 20 bp to 180 bp, from25 bp to 170 bp, from 30 bp to 160 bp, or from 50 bp to 150 bp, e.g.,from 10 bp to 15 bp, from 15 bp to 20 bp, from 20 bp to 25 bp, from 25bp to 30 bp, from 30 bp to 35 bp, from 35 bp to 40 bp, from 40 bp to 45bp, from 45 bp to 50 bp, from 50 bp to 55 bp, from 55 bp to 60 bp, from60 bp to 65 bp, from 65 bp to 70 bp, from 70 bp to 80 bp, from 80 bp to90 bp, from 90 bp to 100 bp, from 100 bp to 150 bp, from 150 bp to 200bp, from 200 bp to 300 bp, from 300 bp to 400 bp, or from 400 bp to 500bp, e.g., 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp,19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29bp, 30 bp, 31 bp, 32 bp, 33 bp, 34 bp, 35 bp, 36 bp, 37 bp, 38 bp, 39bp, 40 bp, 41 bp, 42 bp, 43 bp, 44 bp, 45 bp, 46 bp, 47 bp, 48 bp, 49bp, 50 bp, 51 bp, 52 bp, 53 bp, 54 bp, 55 bp, 56 bp, 57 bp, 58 bp, 59bp, 60 bp, 61 bp, 62 bp, 63 bp, 64 bp, 65 bp, 66 bp, 67 bp, 68 bp, 69bp, 70 bp, 71 bp, 72 bp, 73 bp, 74 bp, 75 bp, 76 bp, 77 bp, 78 bp, 79bp, 80 bp, 81 bp, 82 bp, 83 bp, 84 bp, 85 bp, 86 bp, 87 bp, 88 bp, 89bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99bp, 100 bp, 101 bp, 102 bp, 103 bp, 104 bp, 105 bp, 106 bp, 107 bp, 108bp, 109 bp, 110 bp, 111 bp, 112 bp, 113 bp, 114 bp, 115 bp, 116 bp, 117bp, 118 bp, 119 bp, 120 bp, 121 bp, 122 bp, 123 bp, 124 bp, 125 bp, 126bp, 127 bp, 128 bp, 129 bp, 130 bp, 131 bp, 132 bp, 133 bp, 134 bp, 135bp, 136 bp, 137 bp, 138 bp, 139 bp, 140 bp, 141 bp, 142 bp, 143 bp, 144bp, 145 bp, 146 bp, 147 bp, 148 bp, 149 bp, 150 bp, or more). In someembodiments, the DNA vector includes a DD element).

In another aspect, the invention features an isolated linear DNAmolecule including a plurality of identical amplicons, wherein each ofthe plurality of identical amplicons includes a heterologous gene,wherein the DNA molecule: (a) comprises a terminal repeat sequence(e.g., any of the aforementioned terminal repeat sequences); and (b)lacks an origin of replication and/or a drug resistance gene.

In some embodiments, the circular DNA vector further includes aheterologous gene (e.g., one or more heterologous genes). In someembodiments, the one or more heterologous genes are greater than 4.5 Kbin length (e.g., the one or more heterologous genes, together or eachalone, are from 4.5 Kb to 25 Kb, from 4.6 Kb to 24 Kb, from 4.7 Kb to 23Kb, from 4.8 Kb to 22 Kb, from 4.9 Kb to 21 Kb, from 5.0 Kb to 20 Kb,from 5.5 Kb to 18 Kb, from 6.0 Kb to 17 Kb, from 6.5 Kb to 16 Kb, from7.0 Kb to 15 Kb, from 7.5 Kb to 14 Kb, from 8.0 Kb to 13 Kb, from 8.5 Kbto 12.5 Kb, from 9.0 Kb to 12.0 Kb, from 9.5 Kb to 11.5 Kb, or from 10.0Kb to 11.0 Kb in length, e.g., from 4.5 Kb to 8 Kb, from 8 Kb to 10 Kb,from 10 Kb to 15 Kb, from 15 Kb to 20 Kb in length, or greater, e.g.,from 4.5 Kb to 5.0 Kb, from 5.0 Kb to 5.5 Kb, from 5.5 Kb to 6.0 Kb,from 6.0 Kb to 6.5 Kb, from 6.5 Kb to 7.0 Kb, from 7.0 Kb to 7.5 Kb,from 7.5 Kb to 8.0 Kb, from 8.0 Kb to 8.5 Kb, from 8.5 Kb to 9.0 Kb,from 9.0 Kb to 9.5 Kb, from 9.5 Kb to 10 Kb, from 10 Kb to 10.5 Kb, from10.5 Kb to 11 Kb, from 11 Kb to 11.5 Kb, from 11.5 Kb to 12 Kb, from 12Kb to 12.5 Kb, from 12.5 Kb to 13 Kb, from 13 Kb to 13.5 Kb, from 13.5Kb to 14 Kb, from 14 Kb to 14.5 Kb, from 14.5 Kb to 15 Kb, from 15 Kb to15.5 Kb, from 15.5 Kb to 16 Kb, from 16 Kb to 16.5 Kb, from 16.5 Kb to17 Kb, from 17 Kb to 17.5 Kb, from 17.5 Kb to 18 Kb, from 18 Kb to 18.5Kb, from 18.5 Kb to 19 Kb, from 19 Kb to 19.5 Kb, from 19.5 Kb to 20 Kb,from 20 Kb to 21 Kb, from 21 Kb to 22 Kb, from 22 Kb to 23 Kb, from 23Kb to 24 Kb, from 24 Kb to 25 Kb in length, or greater, e.g., about 4.5Kb, about 5.0 Kb, about 5.5 Kb, about 6.0 Kb, about 6.5 Kb, about 7.0Kb, about 7.5 Kb, about 8.0 Kb, about 8.5 Kb, about 9.0 Kb, about 9.5Kb, about 10 Kb, about 11 Kb, about 12 Kb, about 13 Kb, about 14 Kb,about 15 Kb, about 16 Kb, about 17 Kb, about 18 Kb, about 19 Kb, about20 Kb in length, or greater).

In embodiments of circular DNA vectors having two or more heterologousgenes, the heterologous genes may be the same gene or different genes(e.g., they may encode peptides that interact functionally (e.g., aspart of a signaling pathway) or structurally (e.g., throughdimerization, e.g., a heavy and light chain of an antibody or fragmentthereof)).

In some embodiments, the heterologous gene of the circular DNA vectorincludes one or more trans-splicing molecules or a portion thereof(e.g., a binding domain).

In some embodiments, the circular DNA vector is a monomeric circularvector, a dimeric circular vector, a trimeric circular vector, etc. Insome embodiments, the DNA vector is a monomeric circular vector. In someembodiments, the circular DNA vector (e.g., monomeric circular vector)is double stranded. In some embodiments, the circular DNA vector issupercoiled (e.g., monomeric supercoiled).

In some embodiments, the circular DNA vector includes a promotersequence upstream of the one or more heterologous genes. Additionally,or alternatively, the circular DNA vector can include a polyadenylationsite downstream of the one or more heterologous genes. Thus, in someembodiments, the circular DNA vector includes the following elements,operatively linked from 5′ to 3′ or from 3′ to 5′: (i) a promotersequence; (ii) one or more heterologous genes; (iii) a polyadenylationsite; and (iv) a terminal repeat sequence (e.g., one or more terminalrepeat sequences (e.g., one or more inverted terminal repeat (ITR)sequences (e.g., two ITR sequences) or long terminal repeat (LTR)sequences (e.g., two LTR sequences))).

In another aspect, the invention features methods of producing anisolated circular DNA vector (e.g., any of the circular DNA vectorsdescribed herein). The method includes: (i) providing a sample includinga circular DNA molecule including an AAV genome (e.g., a recombinant AAV(rAAV) genome, e.g., an AAV episome), wherein the AAV genome includes aheterologous gene and a terminal repeat sequence (e.g., one or moreterminal repeat sequences (e.g., one or more inverted terminal repeat(ITR) sequences (e.g., two ITR sequences) or long terminal repeat (LTR)sequences (e.g., two LTR sequences))); (ii) amplifying the AAV genomeusing polymerase (e.g., phage-polymerase)-mediated rolling-circleamplification (e.g., an isothermal polymerase (e.g., phagepolymerase)-mediated rolling circle amplification) to generate a linearconcatamer; (iii) digesting the concatamer using a restriction enzyme togenerate an AAV genome; and (iv) allowing the AAV genome to self-ligateto produce an isolated DNA vector including the heterologous gene andthe terminal repeat sequence. In some embodiments, the method furtherincludes column purifying the isolated DNA vector to purify supercoiledDNA from the isolated DNA vector. The supercoiled DNA can be monomericsupercoiled DNA. In some embodiments, open relaxed circular DNA isseparated from supercoiled DNA in the column purification and can bediscarded. In some embodiments, the heterologous gene is any of theheterologous genes described in any previous aspect, e.g., aheterologous gene that encodes a therapeutic protein configured to treata retinal dystrophy (e.g., a Mendelian-heritable retinal dystrophy, aretinal dystrophy selected from the group consisting of LCA, StargardtDisease, pseudoxanthoma elasticum, rod cone dystrophy, exudativevitreoretinopathy, Joubert Syndrome, CSNB-1C, retinitis pigmentosa, agerelated macular degeneration (AMD), stickler syndrome, microcephaly andchorioretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, andWagner syndrome; a heterologous gene that includes one or more of thefollowing: ABCA4, CEP290, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, IFT-172,C3, COL11A1, TUBGCP6, KIAA1549, CACNA1F, MYO7A, VCAN, USH2A, and HMCN1;a heterologous gene that encodes antibody or portion thereof, acoagulation factor, an enzyme, a growth factor, a hormone, aninterleukin, an interferon, an anti-apoptosis factor, an anti-tumorfactor, a cytokine, and an anti-diabetic factor; and/or a heterologousgene that is a trans-splicing molecule or a portion thereof (e.g., abinding domain).

The polymerase can be a thermophilic polymerase or a polymerase havinghigh processivity through GC-rich residues (e.g., compared to areference polymerase). In some embodiments, the polymerase is a phagepolymerase. In some embodiments, the phage polymerase is Phi29 DNApolymerase.

In another aspect, the invention provides a method of producing anisolated circular DNA vector, the method including: (i) providing asample including a circular DNA molecule including an AAV genome (e.g.,an AAV episome), wherein the AAV genome includes a heterologous gene anda DD element; (ii) amplifying the AAV genome using a firstpolymerase-mediated rolling-circle amplification (e.g., an isothermalpolymerase-mediated rolling circle amplification) to generate a firstlinear concatamer; (iii) digesting the first linear concatamer using arestriction enzyme to generate a first AAV genome; (iv) cloning thefirst AAV genome into a plasmid vector; (v) identifying a plasmid cloneincluding a terminal repeat sequence (e.g., one or more terminal repeatsequences (e.g., one or more inverted terminal repeat (ITR) sequences(e.g., two ITR sequences) or long terminal repeat (LTR) sequences (e.g.,two LTR sequences))); (vi) digesting the plasmid clone including theterminal repeat sequence to generate a second AAV genome; (vii) allowingthe second AAV genome to self-ligate to produce a circular DNA template;(viii) amplifying the circular DNA template using secondpolymerase-mediated rolling-circle amplification (e.g., an isothermalpolymerase-mediated rolling circle amplification) to generate a secondlinear concatamer; (ix) digesting the second linear concatamer using arestriction enzyme to generate a third AAV genome; and (x) allowing thethird AAV genome to self-ligate to produce an isolated DNA vectorincluding the heterologous gene and the terminal repeat sequence. Insome embodiments, the polymerase used in the methods of producingcircular DNA vectors is a phage polymerase (e.g., Phi29 DNA polymerase).

In another aspect, the invention features cell-free methods of producinga therapeutic circular DNA vector, the method including: (i) providing asample including a circular DNA molecule including an AAV genome (e.g.,a recombinant AAV (rAAV) genome, e.g., an AAV episome), wherein the AAVgenome includes a heterologous gene and a terminal repeat sequence(e.g., one or more terminal repeat sequences (e.g., one or more invertedterminal repeat (ITR) sequences (e.g., two ITR sequences) or longterminal repeat (LTR) sequences (e.g., two LTR sequences))); (ii)amplifying the AAV genome using polymerase-mediated rolling-circleamplification (e.g., an isothermal polymerase-mediated rolling circleamplification) to generate a linear concatamer; (iii) digesting theconcatamer using a restriction enzyme to generate an AAV genome; and(iv) allowing the AAV genome to self-ligate to produce an isolatedcircular DNA vector including the heterologous gene and the terminalrepeat sequence. In some embodiments, the polymerase is a phagepolymerase (e.g., Phi29 DNA polymerase). In some embodiments, the methodfurther includes column purifying the isolated DNA vector to purifysupercoiled DNA from the isolated DNA vector. The supercoiled DNA can bemonomeric supercoiled DNA. In some embodiments, open relaxed circularDNA is separated from supercoiled DNA in the column purification and canbe discarded.

In another aspect, provided herein is a pharmaceutical compositionincluding any one or more of the aforementioned circular DNA vectors anda pharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition is non-immunogenic (e.g., substantiallydevoid of bacterial components, such as bacterial signatures, e.g., CpGmotifs). In some embodiments, the pharmaceutical composition issubstantially devoid of viral particles.

In another aspect, the invention features a method of inducingexpression (e.g., episomal expression) of a heterologous gene in asubject in need thereof, the method including administering to thesubject a pharmaceutical composition including any of the aforementionedcircular DNA vectors and a pharmaceutically acceptable carrier (e.g., anon-immunogenic pharmaceutical composition).

In yet another aspect, the invention features methods of treatment usingthe circular DNA vectors and compositions described herein (e.g., any ofthe circular DNA vectors or compositions thereof of the precedingaspects). The invention includes a method of treating a disorder in asubject (e.g., an ocular disorder, e.g., a retinal dystrophy, e.g., aMendelian-heritable retinal dystrophy), the method includingadministering to the subject a pharmaceutical composition of any of thepreceding aspects in a therapeutically effective amount. In someembodiments, the pharmaceutical composition is administered repeatedly(e.g., about twice per day, about once per day, about five times perweek, about four times per week, about three times per week, about twiceper week, about once per week, about twice per month, about once permonth, about once every six weeks, about once every two months, aboutonce every three months, about once every four months, about twice peryear, about once yearly, or less frequently).

In some embodiments, the pharmaceutical composition is administeredlocally (e.g., ocularly, (e.g., intravitreally), intrahepatic,intracerebral, intramuscular, by aerosolization, intradermal,transdermal, or subcutaneous). In some embodiments, the subject is beingtreated for leber's congenital amaurosis (LCA), Stargardt Disease,pseudoxanthoma elasticum, rod cone dystrophy, exudativevitreoretinopathy, Joubert Syndrome, CSNB-1C, age-related maculardegeneration, retinitis pigmentosa, stickler syndrome, microcephaly andchorioretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, orWagner syndrome.

In another aspect, the invention features non-viral isolated DNA vectorsthat replicate the in vivo persistence of rAAV vectors by including adouble D (DD) element in a DNA molecule that is devoid of bacterialplasmid DNA. Thus, the DNA vectors provided herein are non-immunogenicand are not limited to the AAV packaging capacity of about 4.5 Kb. Theinvention also features methods of producing the DD-containing DNAvector, pharmaceutical compositions including the DD-containing DNAvector, and methods of using the vectors described herein, e.g., forinducing episomal expression of a heterologous gene and for treating adisease associated with a defective gene.

In one aspect, the invention provides an isolated DNA vector including aDD element, wherein the DNA vector lacks an origin of replication (e.g.,a bacterial origin of replication) and/or a drug-resistance gene (e.g.,as part of a bacterial plasmid). For example, an isolated DNA vectorincluding a DD element may lack an origin of replication (e.g., abacterial origin of replication). Additionally, or alternatively, anisolated DNA vector including a DD element may lack a drug-resistancegene (e.g., as part of a bacterial plasmid). In some embodiments, anisolated DNA vector including a DD element may lack an origin ofreplication (e.g., a bacterial origin of replication) and adrug-resistance gene (e.g., as part of a bacterial plasmid). In someembodiments, the DNA molecule lacks bacterial plasmid DNA. In someembodiments, the DNA vector lacks an immunogenic bacterial signature(e.g., one or more bacterial-associated CpG motifs, e.g., unmethylatedCpG motifs). In some embodiments, the DNA vector lacks an RNA polymerasearrest site (e.g., an RNA polymerase II (RNAPII) arrest site).

In another aspect, the invention features an isolated DNA vectorincluding a DD element and a bacterial origin of replication and/or adrug resistance gene (e.g., as part of a bacterial plasmid).

In some embodiments of either of the previous aspects, the DNA vectorfurther includes a heterologous gene (e.g., one or more heterologousgenes). In some embodiments, the one or more heterologous genes aregreater than 4.5 Kb in length (e.g., the one or more heterologous genes,together or each alone, are from 4.5 Kb to 25 Kb, from 4.6 Kb to 24 Kb,from 4.7 Kb to 23 Kb, from 4.8 Kb to 22 Kb, from 4.9 Kb to 21 Kb, from5.0 Kb to 20 Kb, from 5.5 Kb to 18 Kb, from 6.0 Kb to 17 Kb, from 6.5 Kbto 16 Kb, from 7.0 Kb to 15 Kb, from 7.5 Kb to 14 Kb, from 8.0 Kb to 13Kb, from 8.5 Kb to 12.5 Kb, from 9.0 Kb to 12.0 Kb, from 9.5 Kb to 11.5Kb, or from 10.0 Kb to 11.0 Kb in length, e.g., from 4.5 Kb to 8 Kb,from 8 Kb to 10 Kb, from 10 Kb to 15 Kb, from 15 Kb to 20 Kb in length,or greater, e.g., from 4.5 Kb to 5.0 Kb, from 5.0 Kb to 5.5 Kb, from 5.5Kb to 6.0 Kb, from 6.0 Kb to 6.5 Kb, from 6.5 Kb to 7.0 Kb, from 7.0 Kbto 7.5 Kb, from 7.5 Kb to 8.0 Kb, from 8.0 Kb to 8.5 Kb, from 8.5 Kb to9.0 Kb, from 9.0 Kb to 9.5 Kb, from 9.5 Kb to 10 Kb, from 10 Kb to 10.5Kb, from 10.5 Kb to 11 Kb, from 11 Kb to 11.5 Kb, from 11.5 Kb to 12 Kb,from 12 Kb to 12.5 Kb, from 12.5 Kb to 13 Kb, from 13 Kb to 13.5 Kb,from 13.5 Kb to 14 Kb, from 14 Kb to 14.5 Kb, from 14.5 Kb to 15 Kb,from 15 Kb to 15.5 Kb, from 15.5 Kb to 16 Kb, from 16 Kb to 16.5 Kb,from 16.5 Kb to 17 Kb, from 17 Kb to 17.5 Kb, from 17.5 Kb to 18 Kb,from 18 Kb to 18.5 Kb, from 18.5 Kb to 19 Kb, from 19 Kb to 19.5 Kb,from 19.5 Kb to 20 Kb, from 20 Kb to 21 Kb, from 21 Kb to 22 Kb, from 22Kb to 23 Kb, from 23 Kb to 24 Kb, from 24 Kb to 25 Kb in length, orgreater, e.g., about 4.5 Kb, about 5.0 Kb, about 5.5 Kb, about 6.0 Kb,about 6.5 Kb, about 7.0 Kb, about 7.5 Kb, about 8.0 Kb, about 8.5 Kb,about 9.0 Kb, about 9.5 Kb, about 10 Kb, about 11 Kb, about 12 Kb, about13 Kb, about 14 Kb, about 15 Kb, about 16 Kb, about 17 Kb, about 18 Kb,about 19 Kb, about 20 Kb in length, or greater).

In embodiments having two or more heterologous genes, the heterologousgenes may be the same gene or different genes (e.g., they may encodepeptides that interact functionally (e.g., as part of a signalingpathway) or structurally (e.g., through dimerization, e.g., a heavy andlight chain of an antibody or fragment thereof)).

In some embodiments, the heterologous gene includes one or moretrans-splicing molecules or portions thereof (e.g., a binding domain).

In some embodiments, the DNA vector is a circular vector (e.g., amonomeric circular vector, a dimeric circular vector, a trimericcircular vector, etc.). In some embodiments, the DNA vector is amonomeric circular vector.

In some embodiments, the DNA vector includes a promoter sequenceupstream of the one or more heterologous genes. Additionally, oralternatively, the DNA vector can include a polyadenylation sitedownstream of the one or more heterologous genes. Thus, in someembodiments, the DNA vector includes the following elements, operativelylinked from 5′ to 3′ or from 3′ to 5′: (i) a promoter sequence; (ii) oneor more heterologous genes; (iii) a polyadenylation site; and (iv) a DDelement.

In another aspect, the invention features methods of producing anisolated DNA vector (e.g., any of the DNA vectors described herein), themethod including: (i) providing a sample including a circular DNAmolecule including an AAV genome (e.g., a recombinant AAV (rAAV) genome,e.g., an AAV episome), wherein the AAV genome includes a heterologousgene and a DD element; (ii) amplifying the AAV genome using polymerase(e.g., phage-polymerase)-mediated rolling-circle amplification (e.g., anisothermal polymerase (e.g., phage polymerase)-mediated rolling circleamplification) to generate a linear concatamer; (iii) digesting theconcatamer using a restriction enzyme to generate an AAV genome; and(iv) allowing the AAV genome to self-ligate to produce an isolated DNAvector including the heterologous gene and the DD element. Thepolymerase can be a thermophilic polymerase or a polymerase having highprocessivity through GC-rich residues (e.g., compared to a referencepolymerase). In some embodiments, the polymerase is a phage polymerase.In some embodiments, the phage polymerase is Phi29 DNA polymerase.

In another aspect, the invention provides a method of producing anisolated DNA vector, the method including: (i) providing a sampleincluding a circular DNA molecule including an AAV genome (e.g., an AAVepisome), wherein the AAV genome includes a heterologous gene and a DDelement; (ii) amplifying the AAV genome using a firstpolymerase-mediated rolling-circle amplification (e.g., an isothermalpolymerase-mediated rolling circle amplification) to generate a firstlinear concatamer; (iii) digesting the first linear concatamer using arestriction enzyme to generate a first AAV genome; (iv) cloning thefirst AAV genome into a plasmid vector; (v) identifying a plasmid cloneincluding a DD element; (vi) digesting the plasmid clone including theDD element to generate a second AAV genome; (vii) allowing the secondAAV genome to self-ligate to produce a circular DNA template; (viii)amplifying the circular DNA template using second polymerase-mediatedrolling-circle amplification (e.g., an isothermal polymerase-mediatedrolling circle amplification) to generate a second linear concatamer;(ix) digesting the second linear concatamer using a restriction enzymeto generate a third AAV genome; and (x) allowing the third AAV genome toself-ligate to produce an isolated DNA vector including the heterologousgene and the DD element. In some embodiments, the polymerase is a phagepolymerase (e.g., Phi29 DNA polymerase).

In another aspect, the invention features cell-free methods of producinga therapeutic DNA vector, the method including: (i) providing a sampleincluding a circular DNA molecule including an AAV genome (e.g., arecombinant AAV (rAAV) genome, e.g., an AAV episome), wherein the AAVgenome includes a heterologous gene and a DD element; (ii) amplifyingthe AAV genome using polymerase-mediated rolling-circle amplification(e.g., an isothermal polymerase-mediated rolling circle amplification)to generate a linear concatamer; (iii) digesting the concatamer using arestriction enzyme to generate an AAV genome; and (iv) allowing the AAVgenome to self-ligate to produce an isolated DNA vector including theheterologous gene and the DD element. In some embodiments, thepolymerase is a phage polymerase (e.g., Phi29 DNA polymerase).

In another aspect, provided herein is a pharmaceutical compositionincluding the DNA vector of any of the preceding aspects and apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition is non-immunogenic (e.g., substantiallydevoid of immunogenic components, such as bacterial signatures, e.g.,CpG motifs). In some embodiments, the pharmaceutical composition issubstantially devoid of viral particles.

In another aspect, the invention features a method of inducingexpression (e.g., episomal expression) of a heterologous gene in asubject in need thereof, the method including administering to thesubject a pharmaceutical composition including the DNA vector of any ofthe preceding aspects and a pharmaceutically acceptable carrier (e.g., anon-immunogenic pharmaceutical composition). In some embodiments, theexpression is induced in the liver of the subject. The liver can secretea therapeutic protein encoded by the heterologous gene (e.g., into theblood).

In yet another aspect, the invention features methods of treatment usingthe DNA vectors and compositions described herein (e.g., any of thevectors or compositions of the preceding aspects). The inventionincludes a method of treating a disorder in a subject (e.g., an oculardisorder, e.g., a retinal dystrophy, e.g., a Mendelian-heritable retinaldystrophy), the method including administering to the subject apharmaceutical composition of any of the preceding aspects in atherapeutically effective amount. In some embodiments, thepharmaceutical composition is administered repeatedly (e.g., about twiceper day, about once per day, about five times per week, about four timesper week, about three times per week, about twice per week, about onceper week, about twice per month, about once per month, about once everysix weeks, about once every two months, about once every three months,about once every four months, about twice per year, about once yearly,or less frequently).

In some embodiments, the pharmaceutical composition is administeredlocally (e.g., ocularly, (e.g., intravitreally), intrahepatic,intracerebral, intramuscular, by aerosolization, intradermal,transdermal, or subcutaneous). In other embodiments, the pharmaceuticalcomposition is administered systemically (e.g., intravenously). In someembodiments, the subject is being treated for leber's congenitalamaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum, rod conedystrophy, exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C,age-related macular degeneration, retinitis pigmentosa, sticklersyndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB2, Usher syndrome, or Wagner syndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the formation of a terminal repeatsequence (in this case, a double D (DD) element) of AAV2. AAV2 invertedterminal repeats (ITRs) are 145-bp in length and located at each end ofthe AAV genome. The ITR contains inverted sequences (designated as A, B,C, and D) that can base-pair and form a hairpin-like structure. A singleITR contains two “A”, “B”, and “C” regions, and a single “D” region. TwoITRs can recombine to form a DD element that is 165 bp in length and issimilar to a single ITR but now contains two “D” regions.

FIGS. 2A-2I are a series of illustrations showing exemplary ITRsequences for various AAV serotypes, showing locations and sequences ofA, B, C, and D elements within an ITR. FIG. 2A is an illustration of anAAV1 ITR. FIG. 2B is an illustration of an AAV2 ITR. FIG. 2C is anillustration of an AAV3 ITR. FIG. 2D is an illustration of an AAV4 ITR.FIG. 2E is an illustration of an AAV5 ITR. FIG. 2F is an illustration ofan AAV6 ITR. FIG. 2G is an illustration of an AAV7 ITR. FIG. 2H is anillustration of a partial AAV8 ITR. FIG. 2I is an illustration of apartial AAV9 ITR.

FIG. 3A is a flow-chart showing exemplary steps of DD vector productionand characterization process described in the Examples. The first stepis to generate or obtain a viral rAAV vector that contains an expressioncassette (e.g., heterologous gene) needed for downstream function. Thevirus infects cells in vitro and forms a circular, double-strandedepisome with a DD element. In the second major step, the circular rAAVgenome is cloned from the cells and sequenced to confirm presence of aDD element. This can then be used to generate a plasmid-based templatefor in vitro DD vector production using rolling circle amplification(steps 3 and 4). The final step is to confirm DD vector gene expressionin vitro before proceeding with in vivo studies.

FIG. 3B is a flow-chart showing exemplary steps of synthetic circularvector production and characterization process described in theExamples. The first step is to generate or obtain a viral rAAV vectorthat contains an expression cassette (e.g., heterologous gene) neededfor downstream function. The virus infects cells in vitro and forms acircular, double-stranded episome with a terminal repeat sequence (inthis case, a DD element). In the second major step, the circular rAAVgenome is cloned from the cells and sequenced to confirm presence of aDD element. This can then be used to generate a plasmid-based templatefor in vitro DD vector production using rolling circle amplification(steps 3 and 4). The final step is to confirm DD vector gene expressionin vitro before proceeding with in vivo studies.

FIG. 4 is a schematic diagram showing a process for generating circularrAAV genomes in vitro. A plasmid with a rAAV genome of interest istransfected with additional AAV production plasmids (tripletransfection) to produce a rAAV viral vector (serotype 2) that containsthe packaged genome. The resulting virus infects HEK293T cells, in whichcircular rAAV genomes are produced.

FIG. 5 is a schematic diagram showing a rolling-circle amplificationreaction for detection of rAAV circular genomes. Total cellular DNA wasdigested with a restriction enzyme that does not cut within the AAVgenome (in this case AvrII). The DNA was then treated with Plasmid-SafeDNase that degrades linear fragments but leaves circular,double-stranded DNA intact. The digestion reaction served as a templatefor linear rolling-circle amplification using random primers and Phi29DNA polymerase. Large, linear concatameric arrays were producedfollowing amplification of circular AAV episomes. The linear arrays weresubsequently digested into unit-length monomeric AAV genomes byrestriction enzyme digestion with EcoRI, which cleaves the AAV genome ata single point. The unit-length AAV genome was then cloned into thepBlueScript vector for further sequence analysis.

FIGS. 6A-6J is a series of illustrations showing exemplary sequences ofvarious AAV2 terminal repeat sequences (in this case, DD elements). FIG.6A is an illustration of a standard DD element including, operativelylinked in a 5′-to-3′ configuration, a 5′ D element, a 5′ A element, a 5′C element, a 3′ C element, a 5′ B element, a 3′ B element, a 3′ Aelement, and a 3′ D element (SEQ ID NO: 9). FIG. 6B is an illustrationof a standard DD element including, operatively linked in a 5′-to-3′configuration, a 5′ D element, a 5′ A element, a 5′ B element, a 3′ Belement, a 5′ C element, a 3′ C element, a 3′ A element, and a 3′ Delement (SEQ ID NO: 10). FIG. 6C is an illustration of a DD elementwithout B elements including, operatively linked in a 5′-to-3′configuration, a 5′ D element, a 5′ A element, a 5′ C element, a 3′ Celement, a 3′ A element, and a 3′ D element (SEQ ID NO: 11). FIG. 6D isan illustration of a DD element without C elements including,operatively linked in a 5′-to-3′ configuration, a 5′ D element, a 5′ Aelement, a 5′ B element, a 3′ B element, a 3′ A element, and a 3′ Delement (SEQ ID NO: 12). FIG. 6E is an illustration of a DD elementwithout B and C elements including, operatively linked in a 5′-to-3′configuration, a 5′ D element, a 5′ A element, a 3′ A element, and a 3′D element (SEQ ID NO: 13). FIG. 6F is an illustration of a DD elementwithout A, B, and C elements including, operatively linked in a 5′-to-3′configuration, a 5′ D element and a 3′ D element (SEQ ID NO: 14). FIG.6G is an illustration of a DD element including, operatively linked in a5′-to-3′ configuration, a 5′ D element, a 5′ A element, a 5′ C element,a nucleic acid sequence in place of a 3′A element, and a 3′ D element(SEQ ID NO: 15). FIG. 6H is an illustration of a DD element including,operatively linked in a 5′-to-3′ configuration, a 5′ D element, a 5′ Aelement, an overlapped 5′ C element with a 3′ A element, and a 3′ Delement (SEQ ID NO: 16). FIG. 6I is an illustration of a DD elementincluding, operatively linked in a 5′-to-3′ configuration, a 5′ Delement, a partial 5′ A element, a partial 3′ A element, and a 3′ Delement (SEQ ID NO: 17). FIG. 6J is an illustration of a DD elementincluding, operatively linked in a 5′-to-3′ configuration, a 5′ Delement, a 5′ A element, a partial 3′ A element, and a 3′ D element (SEQID NO: 18).

FIG. 7 is a schematic illustration showing generation of plasmid-derivedcircular template. Plasmid TG-18 is first digested with EcoRI to releasea linear rAAV genome containing a terminal repeat sequence (DD element;represented as a bowtie). The ends of the linear fragment are ligatedtogether to form a double-stranded circle.

FIG. 8 is a photograph of an agarose gel containing bands of DNA atdifferent steps of the template formation process. Lane 1 is the linearDNA fragment released from the pBlueScript vector. This fragmentcontains the CMV promoter, eGFP cDNA, BGHpA, and the terminal repeatsequence (DD element). Lane 2 is the result of self-ligation of thelinear fragment from Lane 1. Multiple DNA forms are present and includecircular and linear DNA of various sizes resulting from the ligation ofone or multiple DNA fragments. Lane 3 shows the DNA remaining aftertreatment with plasmid-safe DNase that degrades linear, but notcircular, DNA.

FIG. 9 is a schematic diagram showing a process for analyzing Phi29fidelity on amplifying the terminal repeat sequence (DD element). Abacteria-derived circular DD vector serves as a template for linearrolling-circle amplification using random primers and Phi29 DNApolymerase. Large, linear concatameric arrays are produced followingamplification of circular AAV episomes. The linear arrays aresubsequently digested by restriction enzyme digestion to evaluate thepresence of the DD element. The SwaI enzyme cuts on either side of theDD element to produce a 244-bp fragment. The AhdI enzyme cuts oncewithin the DD element and digests the concatamers into unit-lengthfragments of 2.1 Kb.

FIG. 10 is a photograph of an agarose gel showing the results of a Swatdigestion of amplified DNA. DNA amplified from either 1 ng or 6 ng ofthe TG-18 plasmid template was digested with SwaI to produce a 244-bpfragment (Lanes 2 and 3, arrow). This is the same size fragment releasedfrom the original TG-18 plasmid vector (Lane 1). Also included is DNAamplified from a plasmid template lacking the DD element (TG-dDD) thatwas produced by removing the DD element from TG-18 using a SwaI digest(Lanes 4 and 5).

FIG. 11 is a photograph of an agarose gel showing AhdI digestion ofamplified DNA. AhdI cuts once with in the DD element. DNA amplified fromeither 1 ng or 6 ng of the TG-18 plasmid template was digested with AhdIto produce a 2.1-kb fragment (Lanes 1 and 2, arrow). Also included isDNA amplified from a plasmid template lacking the DD element (TG-dDD;lanes 3 and 4). This DNA should not be digested with AhdI as it does notcontain the DD element.

FIG. 12A is a schematic diagram showing self-ligation of a bacterialplasmid-derived template. A plasmid having a terminal repeatsequence-containing vector (here, a DD element-containing vector) isfirst digested with EcoRI to release a linear rAAV genome containing aterminal repeat sequence (a DD element) within the gene sequencerepresented as a bowtie. The ends of the linear fragment are ligatedtogether to form a double-stranded circle.

FIG. 12B is a photograph of an agarose gel showing DNA at differentsteps of the template formation process. Lane 1 is the linear DNAfragment released from the pBlueScript vector. This fragment containsthe CMV promoter, eGFP cDNA, BGHpA, and the DD element. Lane 2 is theresult of self-ligation of the linear fragment from Lane 1. Multiple DNAforms are present and includes circular as well as linear DNA of varioussizes resulting from the ligation of one or multiple DNA fragments. Lane3 shows the DNA remaining after treatment with plasmid-safe DNase thatdegrades linear, but not circular, DNA.

FIG. 13A is a schematic diagram showing the production of linearconcatamers by Phi29 polymerase. The bacteria-derived template shown inFIGS. 11A and 11B served as a template for linear RCA using randomprimers and Phi29 DNA polymerase. Large, linear concatameric arrays wereproduced following amplification of circular AAV episomes. The lineararrays were subsequently digested into unit-length monomeric AAV genomesby restriction enzyme digestion with EcoRI.

FIG. 13B is a photograph of an agarose gel showing size fractionateddigested DNA.

FIG. 14A is a schematic drawing of an in vitro-derived rAAV genome thathas been self-ligated from linear form into a circular product.

FIG. 14B is a photograph of an agarose gel showing the resultingmonomeric circular DNA vector illustrated in FIG. 14A. The majority ofthe DNA is monomeric supercoiled circular DNA.

FIG. 15A is a photomicrograph showing GFP fluorescence of cellstransfected with the synthetic vector characterized in FIG. 14B.Fluorescence was detected using a Spectramax MiniMax300 ImagingCytometer.

FIG. 15B is a photomicrograph showing GFP fluorescence of cellstransfected with the original plasmid containing the rAAV genome.Fluorescence was detected using a Spectramax MiniMax300 ImagingCytometer.

FIG. 16 is a photograph of a Western blot showing GFP expression bycells transfected with pBS alone (lane 1), an in vitro-producedTG-18-derived DD vector (lane 2), an in vitro-produced TG-18-derivedvector without the DD element (lane 3), a plasmid-derived TG-18-derivedDD vector (lane 4), and a plasmid-derived TG-18-derived vector withoutthe DD element (lane 5). Bands showing anti-tubulin staining are shownas a control.

FIG. 17 is a schematic diagram showing an exemplary process forproducing synthetic DNA vectors using rolling circle amplification. Thisprocess includes column purification to separate open circle DNAmolecules from supercoiled DNA monomers.

DETAILED DESCRIPTION

The present invention features non-viral DNA vectors that providelong-term transduction of quiescent cells (e.g., post-mitotic cells) ina manner similar to AAV vectors. The invention is based, in part, on thedevelopment of an in vitro (e.g., cell-free) system to syntheticallyproduce circular AAV-like DNA vectors (e.g., DNA vectors containing aterminal repeat sequence, such as a DD element) by isothermalrolling-circle amplification and ligation-mediated circularization (asopposed to bacterial expression and site-specific recombination, forexample). The present methods allow for improved scalability andmanufacturing efficiency in production of circular AAV-like DNA vectors.Moreover, the vectors produced by these methods are designed to overcomemany of the problems associated with plasmid-DNA vectors, e.g., problemsdiscussed in Lu et al., Mol. Ther. 2017, 25(5): 1187-98, which isincorporated herein by reference in its entirety. For example, byeliminating or reducing the presence of CpG islands and/or bacterialplasmid DNA sequences such as RNAPII arrest sites, transcriptionalsilencing can be reduced or eliminated, resulting in increasedpersistence of the heterologous gene. Further, by eliminating thepresence of immunogenic components (e.g., bacterial endotoxin, DNA, orRNA, or bacterial signatures, such as CpG motifs), the risk ofstimulating the host immune system is reduced. Such benefits areespecially advantageous in the treatment of certain disorders, such asretinal dystrophies (e.g., Mendelian-heritable retinal dystrophies).

Thus, the vectors of the present invention include synthetic DNA vectorsthat: (i) are substantially devoid of bacterial plasmid DNA sequences(e.g., RNAPII arrest sites, origins of replication, and/or resistancegenes) and other bacterial signatures (e.g., immunogenic CpG motifs);and/or (ii) can be synthesized and amplified entirely in a test tube(e.g., replication in bacteria is unnecessary, e.g., bacterial originsof replication and bacterial resistance genes are unnecessary). In someembodiments, the vectors contain a DD element characteristics of AAVvectors. The invention allows a target cell (e.g., a retinal cell) to betransduced with a DNA vector having a heterologous gene that behaveslike AAV viral DNA (e.g., having low transcriptional silencing andenhanced persistence), without needing the virus itself.

I. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs and by reference to publishedtexts, which provide one skilled in the art with a general guide to manyof the terms used in the present application. In the event of anyconflicting definitions between those set forth herein and those of areferenced publication, the definition provided herein shall control.

As used herein, the term “circular vector” or “circular DNA vector”refers to a nucleic acid molecule in a circular form. Such circular formis typically capable of being amplified into concatamers by rollingcircle amplification. A linear double-stranded nucleic acid havingconjoined strands at its termini (e.g., covalently conjugated backbones,e.g., by hairpin loops or other structures) is not a circular vector, asused herein. The term “circular DNA vector” is used interchangeableherein with the term “covalently closed and circular DNA vector” (see,e.g., Example 2). A skilled artisan will understand that such circularvectors may include vectors that are covalently closed with supercoilingand complex DNA topology, as is described herein.

As used herein, a “Mendelian-heritable retinal dystrophy” refers to adisorder of the retina that follows a Mendelian inheritance pattern withvariable penetrance (i.e., complete or reduced penetrance). AMendelian-heritable retinal dystrophy may occur as a result of (a)single mutation in one allele (as in a dominant disorder) or (b) asingle mutation in each allele (as in a recessive disorder). Themutation can be a point mutation, an insertion, a deletion, or a splicevariant mutation. Exemplary Mendelian-heritable retinal dystrophiesinclude Leber's congenital amaurosis (LCA), Stargardt Disease,pseudoxanthoma elasticum, rod cone dystrophy, exudativevitreoretinopathy, Joubert Syndrome, CSNB-1C, retinitis pigmentosa,stickler syndrome, microcephaly and chorioretinopathy, retinitispigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome.Mendelian-heritable retinal dystrophies do not include multifactorialdisorders with multiple genetic associations that together thelikelihood of developing the disease, such as age-related maculardegeneration (AMD).

As used herein, the term “recombination site” refers to a nucleic acidsequence that is a product of site-specific recombination, whichincludes a first sequence that corresponds to a portion of a firstrecombinase attachment site and a second sequence that corresponds to aportion of a second recombinase attachment site. One example of a hybridrecombination site is attR, which is a product of site-specificrecombination and includes a first sequence that corresponds to aportion of attP and a second sequence that corresponds to a portion ofattB. Alternatively, recombination sites can be generated from Cre/Loxrecombination. Thus, a vector generated from Cre/Lox recombination(e.g., a vector including a LoxP site) includes a recombination site, asused herein. Other site-specific recombination events that generaterecombination sites involve, e.g., lambda integrase, FLP recombinase,and Kw recombinase. Nucleic acid sequences that result fromnon-site-specific recombination events (e.g., ITR-mediatedintermolecular recombination) are not recombination sites, as definedherein.

As used herein, the term “therapeutic replacement protein” refers to aprotein that is structurally similar to (e.g., structurally identicalto) a protein that is endogenously expressed by a normal (e.g., healthy)individual. A therapeutic replacement protein can be administered to anindividual that suffers from a disorder associated with a dysfunction of(or lack of) the protein to be replaced. In some embodiments, thetherapeutic replacement protein corrects a defect in a protein resultingfrom a mutation (e.g., a point mutation, an insertion mutation, adeletion mutation, or a splice variant mutation) in the gene encodingthe protein. Therapeutic replacement proteins do not includenon-endogenous proteins, such as proteins associated with a pathogen(e.g., as part of a vaccine). Therapeutic replacement proteins mayinclude enzymes, growth factors, hormones, interleukins, interferons,cytokines, anti-apoptosis factors, anti-diabetic factors, coagulationfactors, anti-tumor factors, liver-secreted proteins, or neuroprotectivefactors. In some instances, the therapeutic replacement protein ismonogenic.

As used herein, the term “therapeutic nucleic acid” refers to a nucleicacid that binds to (e.g., hybridizes with) a molecule (e.g., protein ornucleic acid) in the subject to confer its therapeutic effect (i.e.,without necessarily being transcribed or translated). Therapeuticnucleic acids can be DNA or RNA, such as small interfering RNA (siRNA),short hairpin RNA (shRNA), microRNA (miRNA), a CRISPR molecule (e.g.,guide RNA (gRNA)), an oligonucleotide (e.g., an antisenseoligonucleotide), an aptamer, or a DNA vaccine. In some embodiments, thetherapeutic nucleic acid may be a non-inflammatory or a non-immunogenictherapeutic nucleic acid.

As used herein, the term “terminal repeat sequence” refers to a portionof a nucleic acid molecule having a sequence of nucleotides, wherein thesequence is repeated in adjacent portions of a nucleic acid molecule.The sequences may be repeated in the same or reverse direction (e.g.,ABCDABCD or ABCDDCBA, respectively). In some embodiments, for example,terminal repeat sequences can be, or be derived from (e.g., products ofligation of and/or portions of), inverted terminal repeat sequences(ITRs) or long terminal repeat sequences (LTRs). ITR-derived terminalrepeat sequences may have repeated A elements, B, elements, C elements,and/or D elements (wherein A, B, C, and D elements are defined by SEQ IDNOs: 31-37 and depicted in FIGS. 2A-2H). For example, each of FIGS.6A-6J are terminal repeat sequences, and all DD elements (e.g., SEQ IDNOs: 9 or 10) are examples of a terminal repeat sequence. A terminalrepeat sequence can have a structure that results from homologousrecombination (e.g., intermolecular homologous recombination orintramolecular homologous recombination). A single terminal repeatsequence, on its own, does not form a hairpin.

The term “inverted terminal repeat” or “ITR” refers to the stretch ofnucleic acid that exists in AAV and/or recombinant AAV (rAAV) that canform a T-shaped palindromic structure, that is required for completingAAV lytic and latent life cycles, as described in Muzyczka and Berns,Fields Virology 2001, 2: 2327-2359. The terms “double-D element” and “DDelement” are used interchangeably herein and refer to a type of terminalrepeat sequence which is a DNA structure having a 5′ D element (i.e., anucleic acid sequence with at least 80% homology (e.g., 80%, 85%, 90%,95%, or 100% homology) to a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1, 19, 21, 23, 25, 27, 29, 38, and 40)and a 3′ D element (i.e., a nucleic acid sequence with at least 80%homology (e.g., 80%, 85%, 90%, 95%, or 100% homology) with a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 8, 20,22, 24, 26, 28, 30, 39, and 41 on the same strand of nucleic acid. Insome embodiments, a 5′ D element is 100% homologous to the nucleic acidsequence of SEQ ID NO: 1 and/or a 3′ D element is 100% homologous to thenucleic acid sequence of SEQ ID NO: 8. DD element can be generated byjoining two AAV inverted terminal repeats (ITRs) from the same molecule(intramolecular recombination) or different molecules (intermolecularrecombination) by ligation, as shown in FIG. 1 . Such ligation can occurbetween ITRs of any AAV serotype, exemplary structures of which areshown in FIGS. 2A-2I. A DD element contains two D elements on a singlenucleic acid strand, and may include additional elements, such as one ormore A, B, and/or C elements, or portion(s) thereof, operatively linkingthe 3′ end of the 5′ D element with the 5′ end of the 3′ D element. Insome embodiments, no heterologous genes are present between the 3′ endof the 5′ D element and the 5′ end of the 3′ element. The sequences ofexemplary DD elements derived from AAV2 are shown by each of FIGS.6A-6J. DD elements from other AAV serotypes (e.g., AAV1, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, or AAV9) may be used. Representative 5′ and 3′ Delements from AAV serotypes 1-7 are provided below.

TABLE 1 Representative 5’ and 3’ D elements from AAV serotypes 1-7 SEQID Description NO. Sequence 5’ D, AAV1 19 TTACCCCTAGTGATGGAG 3’ D, AAV120 CTCCATCACTAGGGGTAA 5’ D, AAV2  1 AGGAACCCCTAGTGATGGAG 3’ D, AAV2  8CTCCATCACTAGGGGTTCCT 5’ D, AAV3 21 GCCATACCTCTAGTGATGGAG 3’ D, AAV3 22CTCCATCACTAGAGGTATGGC 5’ D, AAV4 23 GGGCAAACCTAGATGATGGAG 3’ D, AAV4 24CTCCATCATCTAGGTTTGCCC 5’ D, AAV5 25 TACAAAACCTCCTTGCTTGAGAGTGTGGCA3’ D, AAV5 26 TGCCACACTCTCAAGCAAGGAGGTTTTGTA 5’ D, AAV6 27AGGAACCCCTAGTGATGGAG 3’ D, AAV6 28 CTCCATCACTAGGGGTTCCT 5’ D, AAV7 29CGCGGTACCCCTAGTGATGGAC 3’ D, AAV7 30 CTCCATCACTAGGGGTACCGCG 5’ D, AAV838 CGCGCTACCCCTAGTGATGGAG 5’ D, AAV8 39 CTCCATCACTAGGGGTAGCGCG5’ D, AAV9 40 CGCGATTACCCCTAGTGATGGAG 5’ D, AAV9 41CTCCATCACTAGGGGTAATCGCG

The term “heterologous gene” refers to a gene that does not naturallyoccur as part of a viral genome. For instance, a heterologous gene canbe a mammalian gene, e.g., a therapeutic gene (e.g., a gene that encodesa therapeutic replacement protein, an antigen-binding protein, etc.),e.g., a mammalian gene that encodes a therapeutic protein. In someembodiments, a heterologous gene encodes a protein or portion thereofthat is defective or absent in the target cell and/or subject (e.g., atherapeutic replacement protein). In some embodiments, the heterologousgene contains one or more exons encoding a protein that is defective orabsent in the target cell and/or subject. For example, in someembodiments, the heterologous gene includes one or more trans-splicingmolecules or portions thereof (e.g., a binding domain), e.g., asdescribed in WO 2017/087900, which is incorporated herein by referencein its entirety. In some embodiments, a heterologous gene includes atherapeutic nucleic acid, such as a therapeutic RNA (e.g., microRNA).

As used herein, a “trans-splicing molecule” has three main elements: (a)a binding domain (e.g., an oligonucleotide, e.g., an antisenseoligonucleotide) that confers specificity by tethering thetrans-splicing molecule to its target gene (e.g., pre-mRNA); (b) asplicing domain (e.g., a splicing domain having a 3′ or 5′ splice site);and (c) a coding sequence configured to be trans-spliced onto the targetgene, which can replace one or more exons in the target gene (e.g., oneor more mutated exons).

The term “promoter” refers to a sequence that regulates transcription ofa heterologous gene operably linked to the promoter. Promoters providethe sequence sufficient to direct transcription and/or recognition sitesfor RNA polymerase and other transcription factors required forefficient transcription and can direct cell-specific expression. Inaddition to the sequence sufficient to direct transcription, a promotersequence of the invention can also include sequences of other regulatoryelements that are involved in modulating transcription (e.g., enhancers,kozak sequences, and introns). Examples of promoters known in the artand useful in the viral vectors described herein include the CMVpromoter, CBA promoter, smCBA promoter, and those promoters derived froman immunoglobulin gene, SV40, or other tissue specific genes. Standardtechniques are known in the art for creating functional promoters bymixing and matching known regulatory elements. “Truncated promoters” mayalso be generated from promoter fragments or by mix and matchingfragments of known regulatory elements; for example the smCBA promoteris a truncated form of the CBA promoter.

As used herein, a vector or composition (e.g., a pharmaceuticalcomposition containing a DNA vector of the invention) is “substantiallydevoid of” an immunogenic component, such as an immunogenic bacterialsignature, if the composition does not elicit a measurable inflammatoryresponse (e.g., a phenotype associated with toll-like receptorsignaling) in a therapeutically relevant dose. Methods for screeningcompositions for presence of immunogenic components include in vitro andin vivo animal assays according to methods known in the art. In someembodiments, a vector or composition that is substantially devoid of animmunogenic component is non-immunogenic.

As used herein, the term “non-immunogenic” means that a vector orcomposition does not elicit a measurable inflammatory response (e.g., aphenotype associated with toll-like receptor signaling) in atherapeutically relevant dose. Methods for screening compositions forpresence of immunogenic components include in vitro and in vivo animalassays according to methods known in the art. For example, a suitable invitro assay for determining whether a vector or composition isnon-immunogenic involves culturing human peripheral blood mononuclearcells (PBMC) or human PBMC-derived myeloid cells (e.g., monocytes) inthe presence of the vector or composition and measuring the amount ofIL-1β, IL-6, and/or IL-12 in the culture after eight hours. If theconcentration of IL-1β, IL-6, and/or IL-12 is not increased in thesample containing the vector or composition, relative to a negativecontrol, the vector or composition is non-immunogenic.

As used herein, “concatamer” refers to a nucleic acid moleculecomprising multiple copies of the same or substantially the same nucleicacid sequences (e.g., subunits) that are typically linked in a series.

As used herein, the term “isolated” means artificially produced. In someembodiments, with respect to a DNA vector, the term “isolated” refers toa DNA vector that is: (i) amplified in vitro (e.g., in a cell-freeenvironment), for example, by rolling-circle amplification or polymerasechain reaction (PCR); (ii) recombinantly produced by molecular cloning;(iii) purified, as by restriction endonuclease cleavage and gelelectrophoretic fractionation, or column chromatography; or (iv)synthesized by, for example, chemical synthesis. An isolated DNA vectoris one which is readily manipulable by recombinant DNA techniqueswell-known in the art. Thus, a nucleotide sequence contained in a vectorin which 5′ and 3′ restriction sites are known or for which polymerasechain reaction (PCR) primer sequences have been disclosed is consideredisolated, but a nucleic acid sequence existing in its native state inits natural host is not. An isolated DNA vector may be substantiallypurified, but need not be.

As used herein, a “vector” refers to a nucleic acid molecule capable ofcarrying a heterologous gene into a target cell in which theheterologous gene can then be replicated, processed, and/or expressed inthe target cell. After a target cell or host cell processes the genomeof the vector (e.g., by generating a DD element), the genome is notconsidered a vector.

As used herein, a “cell-free method” of producing a DNA vector refers toa method that does not rely on containment of any of the DNA within ahost cell, such as a bacterial (e.g., E. coli) host cell, to facilitateany step of the method. For example, a cell-free method occurs withinone or more synthetic containers (e.g., glass or plastic tubes or othercontainers) within appropriate solutions (e.g., buffered solutions), towhich enzymes and other agents may be added to facilitate DNAamplification, modification, and isolation.

As used herein, a “target cell” refers to any cell that expresses atarget gene and which the vector infects or is intended to infect.Vectors can infect target cells that reside in a subject (in situ) ortarget cells in culture. In some embodiments, target cells of theinvention are post-mitotic cells. Target cells include both vertebrateand invertebrate animal cells (and cell lines of animal origin).Representative examples of vertebrate cells include mammalian cells,such as humans, rodents (e.g., rats and mice), and ungulates (e.g.,cows, goats, sheep and swine). Target cells include ocular cells, suchas retinal cells. Alternatively, target cells can be stem cells (e.g.,pluripotent cells (i.e., a cell whose descendants can differentiate intoseveral restricted cell types, such as hematopoietic stem cells or otherstem cells) or totipotent cells (i.e., a cell whose descendants canbecome any cell type in an organism, e.g., embryonic stem cells, andsomatic stem cells e.g., hematopoietic cells)). In yet otherembodiments, target cells include oocytes, eggs, cells of an embryo,zygotes, sperm cells, and somatic (non-stem) mature cells from a varietyof organs or tissues, such as hepatocytes, neural cells, muscle cellsand blood cells (e.g., lymphocytes).

A “host cell” refers to any cell that harbors a DNA vector of interest.A host cell may be used as a recipient of a DNA vector as described bythe disclosure. The term includes the progeny of the original cell whichhas been transfected. Thus, a “host cell” as used herein may refer to acell which has been transfected with a heterologous gene (e.g., by a DNAvector described herein). It is understood that the progeny of a singleparental cell may not necessarily be completely identical in morphologyor in genomic or total DNA complement as the original parent, due tonatural, accidental, or deliberate mutation.

As used herein, the term “subject” includes any mammal in need of themethods of treatment or prophylaxis described herein. In someembodiments, the subject is a human. Other mammals in need of suchtreatment or prophylaxis include dogs, cats, or other domesticatedanimals, horses, livestock, laboratory animals, including non-humanprimates, etc. The subject may be male or female. In one embodiment, thesubject has a disease or disorder caused by a mutation in the targetgene. In another embodiment, the subject is at risk of developing adisease or disorder caused by a mutation in the target gene. In anotherembodiment, the subject has shown clinical signs of a disease ordisorder caused by a mutation in the target gene. The subject may be anyage during which treatment or prophylactic therapy may be beneficial.For example, in some embodiments, the subject is 0-5 years of age, 5-10years of age, 10-20 years of age, 20-30 years of age, 30-50 years ofage, 50-70 years of age, or more than 70 years of age.

As used herein, an “effective amount” or “effective dose” of a vector orcomposition thereof refers to an amount sufficient to achieve a desiredbiological and/or pharmacological effect, e.g., when delivered to a cellor organism according to a selected administration form, route, and/orschedule. As will be appreciated by those of ordinary skill in this art,the absolute amount of a particular vector or composition that iseffective can vary depending on such factors as the desired biologicalor pharmacological endpoint, the agent to be delivered, the targettissue, etc. Those of ordinary skill in the art will further understandthat an “effective amount” can be contacted with cells or administeredto a subject in a single dose or through use of multiple doses.

As used herein, the term “persistence” refers to the duration of timeduring which a gene is expressible in a cell. Persistence of a DNAvector, or persistence of a heterologous gene within a DNA vector, canbe quantified relative to a reference vector, such as a control vectorproduced in bacteria (e.g., a circular vector produced in bacteria orhaving one or more bacterial signatures not present in the vector of theinvention), using any gene expression characterization method known inthe art. In some embodiments, a control vector lacks a DD element.Additionally, or alternatively, persistence can be quantified at anygiven time point following administration of the vector. For example, insome embodiments, a heterologous gene of a DNA vector of the inventionpersists for at least six months after administration if its expressionis detected in situ six months after administration of the vector. Insome embodiments, a gene “persists” in a target cell if itstranscription is detectable at three months, four months, five months,six months, seven months, eight months, nine months, ten months, elevenmonths, one year, two years, or longer after administration. In someembodiments, a gene is said to persist if any detectable fraction of theoriginal expression level remains (e.g., at least 1%, at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 70%, or at least 100% of the original expression level) after agiven period of time after administration (e.g., three months, fourmonths, five months, six months, seven months, eight months, ninemonths, ten months, eleven months, one year, two years, or longer afteradministration).

As used herein, a “mutation” refers to any aberrant nucleic acidsequence that causes a defective (e.g., non-functional, reducedfunction, aberrant function, less than normal amounts produced) proteinproduct. Mutations include base pair mutations (e.g., single nucleotidepolymorphisms), missense mutations, frameshift mutations, deletions,insertions, and splice mutations.

As used herein, the terms “disorder associated with a mutation” or“mutation associated with a disorder” refer to a correlation between adisorder and a mutation. In some embodiments, a disorder associated witha mutation is known or suspected to be wholly or partially, or directlyor indirectly, caused by the mutation. For example, a subject having themutation may be at risk of developing the disorder, and the risk mayadditionally depend on other factors, such as other (e.g., independent)mutations (e.g., in the same or a different gene), or environmentalfactors.

As used herein, the term “immune disorder” refers to a dysfunction ofthe immune system characterized by a compromised (underactive) immunefunction (e.g., an inability to mount a suitable immune response toforeign or pathogenic antigen) or an overactive immune function (e.g.,an inability to distinguish endogenous “self” antigen from foreign orpathogenic antigen, thereby leading to, e.g., aberrant inflammation,chronic infection, and/or autoimmunity). Immune disorders do not includenormal immune responses (e.g., normal inflammation, infection, andpathogen-clearance). Thus, a vaccine, for example, is not a treatmentfor an immune disorder, as defined herein.

As used herein, the term “treatment,” or a grammatical derivationthereof, is defined as reducing the progression of a disease, reducingthe severity of a disease symptom, retarding progression of a diseasesymptom, removing a disease symptom, or delaying onset of a disease.

As used herein, the term “prevention” of a disorder, or a grammaticalderivation thereof, is defined as reducing the risk of onset of adisease, e.g., as a prophylactic therapy for a subject who is at risk ofdeveloping a disorder associated with a mutation. A subject can becharacterized as “at risk” of developing a disorder by identifying amutation associated with the disorder, according to any suitable methodknown in the art or described herein. In some embodiment, a subject whois at risk of developing a disorder has one or more mutations associatedwith the disorder. Additionally, or alternatively, a subject can becharacterized as “at risk” of developing a disorder if the subject has afamily history of the disorder.

The term “administering,” or a grammatical derivation thereof, as usedin the methods described herein, refers to delivering the composition,or an ex vivo-treated cell, to the subject in need thereof, e.g., havinga mutation or defect in the targeted gene. For example, in oneembodiment in which ocular cells are targeted, the method involvesdelivering the composition by subretinal injection to the photoreceptorcells or other ocular cells. In another embodiment, intravitrealinjection to ocular cells or injection via the palpebral vein to ocularcells may be employed. In another embodiment, the composition isadministered intravenously. Still other methods of administration may beselected by one of skill in the art, in view of this disclosure.

The term “pharmaceutically acceptable” means safe for administration toa mammal, such as a human. In some embodiments, a pharmaceuticallyacceptable composition is approved by a regulatory agency of the Federalor a state government or listed in the U. S. Pharmacopeia or othergenerally recognized pharmacopeia for use in animals, and moreparticularly in humans.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which a vector or composition of the invention is administered.Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,2^(nd) edition, 2005.

The terms “a” and “an” mean “one or more of.” For example, “a gene” isunderstood to represent one or more such genes. As such, the terms “a”and “an,” “one or more of a (or an),” and “at least one of a (or an)”are used interchangeably herein.

As used herein, the term “about” refers to a value within ±10%variability from the reference value, unless otherwise specified.

For any conflict in definitions between various sources or references,the definition provided herein shall control.

II. Vectors

Provided herein are synthetic DNA vectors featuring a heterologous gene.The DNA vector may further include a double D (DD) element. SyntheticDNA vectors having DD elements can persist intracellularly (e.g., inquiescent cells, such as post-mitotic cells) as episomes, e.g., in amanner similar to AAV vectors. Vectors provided herein can be naked DNAvectors, devoid of components inherent to viral vectors (e.g., viralproteins) and bacterial plasmid DNA, such as immunogenic components(e.g., immunogenic bacterial signatures (e.g., CpG islands or CpGmotifs)) or components additionally, or otherwise associated withreduced persistence (e.g., CpG islands or CpG motifs).

Further provided are synthetic circular DNA vectors featuring aheterologous gene without an origin of replication and/or a drugresistance gene, herein referred to as circular DNA vectors. The presentinvention provides circular DNA vectors that are produced synthetically.

Synthetic circular DNA vectors of the invention can persistintracellularly (e.g., in quiescent cells, such as post-mitotic cells)as episomes, e.g., in a manner similar to AAV vectors. Vectors providedherein can be naked DNA vectors, devoid of components inherent to viralvectors (e.g., viral proteins) and bacterial plasmid DNA, such asimmunogenic components (e.g., immunogenic bacterial signatures (e.g.,CpG motifs)) or components additionally or otherwise associated withreduced persistence (e.g., CpG islands). For example, in someembodiments, the vector contains DNA in which at least 50% (e.g., atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 97%, at least 99%, or essentially all) of the DNA lacks one ormore elements of bacterial plasmid DNA, such as immunogenic components(e.g., immunogenic bacterial signatures (e.g., CpG motifs)) orcomponents additionally or otherwise associated with reduced persistence(e.g., CpG islands). In some embodiments, at least 50% (e.g., at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least97%, at least 99%, or essentially all) of the DNA lacks CpG methylation.In some embodiments, the vector contains DNA in which at least 50%(e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 97%, at least 99%, or essentially all) of the DNA lacksbacterial methylation signatures, such as Dam methylation and Dcmmethylation. For examples, in some embodiments, the vector contains DNAin which at least 50% (e.g., at least 60%, at least 70%, at least 80%,at least 90%, at least 95%, at least 97%, at least 99%, or essentiallyall) of the GATC sequences are unmethylated (e.g., by Dam methylase).Additionally or alternatively, the vector contains DNA in which at least50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 97%, at least 99%, or essentially all) of the CCAGGsequences and/or CCTGG sequences are unmethylated (e.g., by Dcmmethylase).

In some embodiments regarding each of the aforementioned vectors, theDNA vector is persistent in vivo (e.g., the circularity andnon-bacterial nature (i.e., by in vitro (e.g., cell-free) synthesis) areassociated with long-term transcription or expression of a heterologousgene of the DNA vector). In some embodiments, the persistence of thecircular DNA vector is from 5% to 50% greater, 50% to 100% greater,one-fold to five-fold, or five-fold to ten-fold (e.g., at least 5%, 10%,20%, 30%, 40%, 50%, 75%, one-fold, two-fold, three-fold, four-fold,five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, ormore) greater than a reference vector (e.g., a circular vector producedin bacteria or having one or more bacterial signatures not present inthe vector of the invention). In some embodiments, the circular DNAvector of the invention persists for one week to four weeks, from onemonth to four months, from four months to one year, from one year tofive years, from five years to twenty years, or from twenty years tofifty years (e.g., at least one week, at least two weeks, at least onemonth, at least four months, at least one year, at least two years, atleast five years, at least ten years, at least twenty years, at leastthirty years, at least forty years, or at least fifty years). In someembodiments, the DNA vector includes a DD element, which may beassociated with increased persistence.

A DNA vector may be a circular DNA vector. The circular DNA vector maybe monomeric, dimeric, trimeric, tetrameric, pentameric, hexameric, etc.Preferably, the circular DNA vector is monomeric. In other preferredembodiments, the circular DNA vector is a monomeric, supercoiledcircular DNA molecule. In some embodiments, the DNA vector is nicked. Insome embodiments, the DNA vector is open circular. In some embodiments,the DNA vector is double-stranded circular.

Additionally, or alternatively, the DNA vector may include a DD element.In certain embodiments, the DNA vector (e.g., the circular DNA vector,e.g., the monomeric circular DNA vector) includes, operatively linked inthe 5′ to 3′ direction: (i) a 5′ D element, (ii) a heterologous gene,and (iii) a 3′ D element. In some embodiments, the DNA vector comprises,operatively linked in the 5′ to 3′ direction: (i) a 5′ D element, (ii) apromoter, (iii) a heterologous gene, and (iv) a 3′ D element. In someembodiments, the DNA vector comprises, operatively linked in the 5′ to3′ direction: (i) a 5′ D element, (ii) a promoter, (iii) a heterologousgene, (iv) a polyadenylation site, and (v) a 3′ D element.

For example, a DNA vector may include, operatively linked in a 5′ to 3′direction: (i) a 5′ A element, (ii) 5′ D element, (iii) a heterologousgene, (iv) a 3′ D element, and (v) a 5′ A element. In some embodiments,the DNA vector includes, in a 5′ to 3′ direction: (i) a 5′ A element,(ii) 5′ D element, (iii) a promoter, (iv) a heterologous gene, (v) a 3′D element, and (vi) a 5′ A element. In some embodiments, the DNA vectorincludes, in a 5′ to 3′ direction: (i) a 5′ A element, (ii) 5′ Delement, (iii) a promoter, (iv) a heterologous gene, (v) apolyadenylation site, (vi) a 3′ D element, and (vii) a 5′ A element. Insome embodiments, the DNA vector includes, in a 5′ to 3′ direction: (i)a 5′ C element, (ii) a 5′ A element, (iii) 5′ D element, (iv) aheterologous gene, (v) a 3′ D element, (vi) a 3′ A element, and (vii) a3′ B element. In some embodiments, the DNA vector includes, in a 5′ to3′ direction: (i) a 5′ C element, (ii) a 5′ A element, (iii) 5′ Delement, (iv) a promoter, (v) a heterologous gene, (vi) a 3′ D element,(vii) a 3′ A element, and (viii) a 3′ B element. In some embodiments,the DNA vector includes, in a 5′ to 3′ direction: (i) a 5′ C element,(ii) a 5′ A element, (iii) 5′ D element, (iv) a promoter, (v) aheterologous gene, (vi) a polyadenylation site, (vii) a 3′ D element,(viii) a 3′ A element, and (ix) a 3′ B element.

In some embodiments, the DNA vector includes a DD element having anucleic acid sequence having at least a 5′ D element and a 3′ D elementon the same nucleic acid (e.g., DNA) strand. For example, in someembodiments, the DNA vector includes, operatively linked in a 5′ to 3′direction: (i) a heterologous gene and (ii) a DD element. In someembodiments, the DNA vector includes, in a 5′ to 3′ direction: (i) apromoter, (ii) a heterologous gene, and (iii) DD element. In someembodiments, the DNA vector includes, in a 5′ to 3′ direction: (i) aheterologous gene, (ii) a polyadenylation site, and (iii) a DD element.In some embodiments, the DNA vector includes, in a 5′ to 3′ direction:(i) a promoter, (ii) a heterologous gene, (iii) a polyadenylation site,and (iv) a DD element.

Terminal Repeat Sequences

In some embodiments of the present invention, vectors and compositionsprovided herein include terminal repeat sequences, which may be derived,e.g., from ITRs, LTRs, or other terminal structures, e.g., as a resultof circularization. The terminal repeat sequence can be at least 10 basepairs (bp) in length (e.g., from 10 bp to 500 bp, from 12 bp to 400 bp,from 14 bp to 300 bp, from 16 bp to 250 bp, from 18 bp to 200 bp, from20 bp to 180 bp, from 25 bp to 170 bp, from 30 bp to 160 bp, or from 50bp to 150 bp, e.g., from 10 bp to 15 bp, from 15 bp to 20 bp, from 20 bpto 25 bp, from 25 bp to 30 bp, from 30 bp to 35 bp, from 35 bp to 40 bp,from 40 bp to 45 bp, from 45 bp to 50 bp, from 50 bp to 55 bp, from 55bp to 60 bp, from 60 bp to 65 bp, from 65 bp to 70 bp, from 70 bp to 80bp, from 80 bp to 90 bp, from 90 bp to 100 bp, from 100 bp to 150 bp,from 150 bp to 200 bp, from 200 bp to 300 bp, from 300 bp to 400 bp, orfrom 400 bp to 500 bp, e.g., 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp,16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26bp, 27 bp, 28 bp, 29 bp, 30 bp, 31 bp, 32 bp, 33 bp, 34 bp, 35 bp, 36bp, 37 bp, 38 bp, 39 bp, 40 bp, 41 bp, 42 bp, 43 bp, 44 bp, 45 bp, 46bp, 47 bp, 48 bp, 49 bp, 50 bp, 51 bp, 52 bp, 53 bp, 54 bp, 55 bp, 56bp, 57 bp, 58 bp, 59 bp, 60 bp, 61 bp, 62 bp, 63 bp, 64 bp, 65 bp, 66bp, 67 bp, 68 bp, 69 bp, 70 bp, 71 bp, 72 bp, 73 bp, 74 bp, 75 bp, 76bp, 77 bp, 78 bp, 79 bp, 80 bp, 81 bp, 82 bp, 83 bp, 84 bp, 85 bp, 86bp, 87 bp, 88 bp, 89 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96bp, 97 bp, 98 bp, 99 bp, 100 bp, 101 bp, 102 bp, 103 bp, 104 bp, 105 bp,106 bp, 107 bp, 108 bp, 109 bp, 110 bp, 111 bp, 112 bp, 113 bp, 114 bp,115 bp, 116 bp, 117 bp, 118 bp, 119 bp, 120 bp, 121 bp, 122 bp, 123 bp,124 bp, 125 bp, 126 bp, 127 bp, 128 bp, 129 bp, 130 bp, 131 bp, 132 bp,133 bp, 134 bp, 135 bp, 136 bp, 137 bp, 138 bp, 139 bp, 140 bp, 141 bp,142 bp, 143 bp, 144 bp, 145 bp, 146 bp, 147 bp, 148 bp, 149 bp, 150 bp,or more).

In some embodiments of the present invention, a terminal repeat sequenceof a synthetic vector can be a DD element (e.g., a DD element derivedfrom, and/or containing one or more portions of an ITR). A DD elementcontains two D elements on a single DNA molecule. In some embodiments,the two D elements are separated by about 125 nucleic acids. DD elementscan be derived from an AAV of any serotype, e.g., AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.

In some embodiments, the DD element comprises two D elements directlyjoined to one another, for example, in the configuration shown in FIG.6F. Thus, in some embodiments, the DD element has the nucleic acidsequence of SEQ ID NO: 14. In some embodiments, the DD element is 80%,82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 100% homologous to thenucleic acid sequence of SEQ ID NO: 14.

In other embodiments, a DD element of the present invention has at leastone additional element separating the 5′ D element from the 3′ Delement, such as one or more A elements; one or more B elements; and/orone or more C elements, which may be arranged in any suitable order. Forexample, in some embodiments, the DD element comprises, operativelylinked in a 5′-to-3′ configuration: (i) a 5′ D element (i.e., a nucleicacid sequence having at least 80% homology (e.g., 80%, 85%, 90%, 95%, or100% homology) to the nucleic acid sequence of any one of SEQ ID NOs: 1,19, 21, 23, 25, 27, 29, 38, or 40; (ii) one or more internal nucleicacids (e.g., non-heterologous nucleic acids), and (iii) a 3′ D element(i.e., a nucleic acid sequence having at least 80% homology (e.g., 80%,85%, 90%, 95%, or 100% homology) to the nucleic acid sequence of any oneof SEQ ID NOs: 8, 20, 22, 24, 26, 28, 30, 39, or 41. In someembodiments, the one or more nucleic acids of (ii) is from 1-125 nucleicacids, 2-100 nucleic acids, 5-80 nucleic acids, or 10-50 nucleic acids,e.g., 1-20 nucleic acids, 20-40 nucleic acids, 40-60 nucleic acids,60-80 nucleic acids, 80-100 nucleic acids, or 100-125 nucleic acids,e.g., 1-5 nucleic acids, 5-10 nucleic acids, 10-15 nucleic acids, 15-20nucleic acids, 20-25 nucleic acids, 25-30 nucleic acids, 30-35 nucleicacids, 35-40 nucleic acids, 40-45 nucleic acids, 45-50 nucleic acids,50-55 nucleic acids, 55-60 nucleic acids, 60-65 nucleic acids, 65-70nucleic acids, 70-75 nucleic acids, 75-80 nucleic acids, 80-85 nucleicacids, 85-90 nucleic acids, 90-95 nucleic acids, 95-100 nucleic acids,100-105 nucleic acids, 105-110 nucleic acids, 110-115 nucleic acids,115-120 nucleic acids, 120-125 nucleic acids, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125nucleic acids).

In some embodiments, the DD element comprises two D elements (e.g., a 5′D element (e.g., SEQ ID NO: 1, 19, 21, 23, 25, 27, 29, 38, or 40) and a3′ D element (e.g., SEQ ID NO: 8, 20, 22, 24, 26, 28, 30, 39, or 41)),in addition to two A elements (e.g., a 5′ A element (e.g., SEQ ID NO: 2)and a 3′ A element (e.g., SEQ ID NO: 7)), two B elements (e.g., a 5′ Belement (e.g., SEQ ID NO: 5) and a 3′ B element (e.g., SEQ ID NO: 6)),and two C elements, e.g., SEQ ID NOs: 1-8. The nucleic acid sequences ofSEQ ID NOs: 1-8 may be operatively linked in order in a 5′ to 3′direction, for example, as shown in FIG. 6A. Thus, in some embodiments,the DD element comprises the nucleic acid sequence of SEQ ID NO: 9.Alternatively, SEQ ID NOs: 1-8 can be operatively linked in any suitableorder. For example, in some embodiments, the DD element comprises thenucleic acid sequence of SEQ ID NO: 10. In particular embodiments, SEQID NOs: 1 and 8 (i.e., the two D elements) flank the remaining elementsand/or nucleic acids within the D element.

The elements of SEQ ID NOs: 1-8 can each be directly linked orindirectly linked (e.g., operatively linked) to one another, e.g., SEQID NOs: 1-8 can be operatively linked in a 5′ to 3′ direction.Alternatively, there may be one or more nucleic acids separating one ormore operatively linked elements, as shown in FIGS. 6A and 6B. In someembodiments, the DD element comprises 1-100 additional nucleic acids(e.g., 3-50 nucleic acids, e.g., 3-10 nucleic acids, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, or more additional nucleic acids) positionedbetween the 5′ D element and the 3′ D element (e.g., between one, two,three, four, five, or more of the following pairs of elements: a 5′ Delement and a 5′ A element, a 5′ D element and a 5′ B element, a 5′ Delement and a 3′ B element, a 5′ D element and a 5′ C element, a 5′ Delement and a 3′ C element, a 5′ D element and a 3′ A element, a 5′ Delement and a 3′ D element, a 5′ A element and a 5′ B element, a 5′ Aelement and a 3′ B element, a 5′ A element and a 5′ C element, a 5′ Aelement and a 3′ C element, a 5′ A element and a 3′ A element, a 5′ Aelement and a 3′ D element, a 5′ B element and a 3′ B element, a 5′ Belement and a 5′ C element, a 5′ B element and a 3′ C element, a 5′ Belement and a 3′ A element, a 5′ B element and a 3′ D element, a 3′ Belement and a 5′ C element, a 3′ B element and a 3′ C element, a 3′ Belement and a 3′ A element, a 3′ B element and a 3′ D element, a 5′ Celement and a 3′ C element, a 5′ C element and a 3′ A element, a 5′ Celement and a 3′ D element, a 3′ C element and a 3′ A element, a 3′ Celement and a 3′ D element, or a 3′ A element and a 3′ D element).

Additional nucleic acids may serve, for example, as restriction sites,as shown by the AhdI sites in FIGS. 6A and 6B.

In some embodiments, one or more of elements A, B, or C (e.g., SEQ IDNOs: 2-7) are absent. For example, FIG. 6C shows a AAV2-derived DDelement without B elements. Thus, in some embodiments, the DD element ofthe invention may have a nucleic acid sequence having 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 11. Similarly,FIG. 6D shows a DD element without C elements. Thus, in someembodiments, the DD element of the invention may have a nucleic acidsequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homologywith SEQ ID NO: 12. In some embodiments, the DD element does notcomprise B or C elements, such as shown in FIG. 6E. Thus, in someembodiments, the DD element of the invention may have a nucleic acidsequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homologywith SEQ ID NO: 13.

Alternatively, one or more of elements A, B, or C (e.g., SEQ ID NOs:2-7) may be replaced by a dissimilar nucleic acid sequence, such as inFIG. 6G, which shows a suitable DD element having a different nucleicacid sequence in place of its 3′ A element. Thus, in some embodiments,the DD element comprises SEQ ID NOs: 1-3 and 8. In some embodiments, theDD element of the invention may have a nucleic acid sequence having 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 15.

In some embodiments, one or more (e.g., one, two, three, four, five,six, or more) nucleic acids overlap between two adjacent elements. Forexample, in some embodiments wherein the 3′-terminal one or more nucleicacids of a first element match the 5-terminal one or more nucleic acidsof a second element linked to its 3′ end, the overlapping nucleic acidsneed not be repeated. An example of such a DD element is shown in FIG.6H, where the 3′ end of the 5′ C element overlaps with the 5′ end of the3′A element. Thus, in some embodiments, the DD element of the inventionmay have a nucleic acid sequence having 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100% homology with SEQ ID NO: 16.

Nucleic acid sequences between the 5′ and 3′ D elements may be portionsof any one or more of the 5′ or 3′A elements, 5′ or 3′ B elements, or 5′or 3′ C elements. In particular embodiments, the DD element comprisesone or more partial A elements, such as shown in FIGS. 6I and 6J. Apartial A element may comprise a nucleic acid sequence having six ormore consecutive matching nucleic acids as SEQ ID NOs: 2 or 7 (e.g.,6-40, 8-35, 10-30, or 15-25, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or 40 consecutive matching nucleic acids). Insome embodiments, the DD element of the invention may have a nucleicacid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%homology with SEQ ID NO: 17. In some embodiments, the DD element of theinvention may have a nucleic acid sequence having 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% homology with SEQ ID NO: 18.

Exemplary nucleic acid sequences of AAV2-derived DD elements andsub-elements thereof are provided in Table 2, below.

TABLE 2Exemplary nucleic acid sequences of DD elements and sub-elements thereofSEQ ID NO: Description Sequence  1 5’ D element AGGAACCCCTAGTGATGGAG  25’ A element TTGGCCACTCCCTCTCTGCGCGCTDGCTCGCTCACTGAGGC  3 5’ C elementCGCCCGGGC  4 3’ C element GCCCGGGCG  5 5’ B element CGGGCGACC  63’ B element GGTCGCCCG  7 3’ A elementGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA  8 3’ D elementCTCCATCACTAGGGGTTCCT  9 StandardAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGC DD (flop)TCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 10 StandardAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGC DD (flip)TCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 11 Deleted BBAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTC CATCACTAGGGGTTCCT 12Deleted CC AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCC ATCACTAGGGGTTCCT 13 DeletedAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGC BBCCTCGCTCGCTCACTGAGGCGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 14 DeletedAGGAACCCCTAGTGATGGAGCTCCATCACTAGGGGTTCCT BBCCAA 15 Clone:AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGC T88-16TCGCTCGCTCACTGAGGCCGCCCGGGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 16 Clone:AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGC 302A-12TCGCTCGCTCACTGAGGCCGCCCGGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CT 17 Clone:AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCGCAGAGAG 304B-68GGAGTGGCCAACTCCATCACTAGGGGTTCCT 18 Clone:AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGC 307A-9TCGCTCGCTCACTGAGGCCGAGCGCGCAGAGAGGGAGTGGCC AACTCCATCACTAGGGGTTCCTHeterologous Genes

Any of the vectors of the present invention (e.g., DNA vectorscontaining a DD element, having a circular structure, or both) can beused to insert a heterologous gene into a target cell. As disclosedherein, a broad range of heterologous genes may be delivered to targetcells by way of the present vectors. In some embodiments, theheterologous gene is configured to transfect a target cell having amutation associated with a disease which can be treated by expression ofthe heterologous gene, e.g., a gene encoding a therapeutic protein,e.g., a protein that is defective or absent in the target cell and/orsubject.

In such instances, the heterologous gene may encode all or a portion of(e.g., as part of a trans-splicing molecule) an ocular protein, such asCEP290, ABCA4, ABCC6, RIMS1, LRP5, CC2D2A, TRPM1, C3, IFT172, COL11A1,TUBGCP6, KIAA1549, CACNA1F, SNRNP200, RP 1, MYO7A, PRPF8, VCAN, USH2A,and HMCN1. Other exemplary therapeutic proteins include one or morepolypeptides selected from the group consisting of growth factors,interleukins, interferons, anti-apoptosis factors, cytokines,anti-diabetic factors, anti-apoptosis agents, coagulation factors,anti-tumor factors, liver-secreted proteins, neuroprotective factors, orneurotrophins. Therapeutic proteins may include BDNF, CNTF, CSF, EGF,FGF, G-SCF, GM-CSF, gonadotropin, IFN, IFN-α, IFN-γ IFG-1, M-CSF, NGF,PDGF, PEDF, TGF, VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAP1, I-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10, viralIL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and/or IL-18.

The heterologous gene may encode all or a portion of a neuroprotectivefactor, such as Kifap3, Bcl-xl, Crmp1, Chk.beta., CALM2, Caly, NPG11,NPT1, Eef1a1, Dhps, Cd151, Morf412, CTGF, LDH-A, Atl1, NPT2, Ehd3,Cox5b, Tuba1a, gamma-actin, Rpsa, NPG3, NPG4, NPG5, NPG6, NPG7, NPG8,NPG9, and NPG10. Exemplary neurotrophins are NGF, BDNF, NT-3, NT-4, andCNTF.

In some instances, the heterologous gene is associated with a disorderselected from the group consisting of an ocular disorder, a liverdisorder, a neurological disorder, an immune disorder, a cancer, acardiovascular disorder, a blood coagulation disorder, a lysosomalstorage disorder, or type 2 diabetes.

Heterologous genes for treatment of blood coagulation disorders includegenes that correct a defect in a coagulation factor or set ofcoagulation factors, such as one or more coagulation factors selectedfrom the group consisting of fibrinogen, prothrombin, thromboplastin,factor V, factor VII (e.g., factor VIIa), factor VIII, factor IX, factorX, factor XI, factor XII, factor XIII, or von Willebrand factor. In someinstances, the heterologous gene encodes for fibrinogen, prothrombin,thromboplastin, factor V, factor VII, factor VIII, factor IX, factor X,factor XI, factor XII, factor XIII, or von Willebrand factor. Forexample, in some instances, a heterologous gene encoding for fibrinogenis FGA, FGB, or FGG; a heterologous gene encoding for prothrombin is F2;a heterologous gene encoding for factor V is F5; a heterologous geneencoding for factor VII is F7; a heterologous gene encoding for factorVIII is F8; a heterologous gene encoding for factor IX is F9; aheterologous gene encoding for factor X is F10; a heterologous geneencoding for factor XII is F11; a heterologous gene encoding for factorXII is F12; a heterologous gene encoding for factor XIII is F13A orF13B. In some instances, the heterologous gene is LMAN1 or MCFD2.Alternatively, the heterologous gene may encode for an enzyme involvedin the posttranslational modifications of any of the precedingcoagulation factors.

Other heterologous genes encoding polypeptides of interest can beincluded as part of the vectors of the invention, including for example,growth hormones to promote growth in a transgenic animal, orinsulin-like growth factors (IGFs), α-anti-trypsin, erythropoietin(EPO), factors VIII, IX, X, and XI of the blood clotting system,LDL-receptor, GATA-1, etc. The nucleic acid sequence may include asuicide gene encoding, e.g., apoptotic or apoptosis-related enzymes andgenes including AIF, Apaf, (e.g., Apaf-1, Apaf-2, or Apaf-3) APO-2 (L),APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2, Bcl-x.sub.L, Bcl-x.sub.S, bik,CAD, Calpain, Caspases e.g. Caspase-1, Caspase-2, Caspase-3, Caspase-4,Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10,Caspase-11, or Granzyme B, ced-3, ced-9, Ceramide, c-Jun, c-Myc, CPP32,crm A, Cytochrome c, D4-GDP-DI, Daxx, CdR1, DcR1, DD, DED, DISC,DNA-PK.sub.CS, DR3, DR4, DR5, FADD/MORT-1, FAK, Fas, Fas-ligand CD95/fas(receptor), FLICE/MACH, FLIP, Fodrin, fos, G-Actin, Gas-2, Gelsolin,glucocorticoid/glucocorticoid receptor, granzyme A/B, hnRNPs C1/C2,ICAD, ICE, JNK, Lamin A/B, MAP, MCL-1, Mdm-2, MEKK-1, MORT-1, NEDD,NF-κB, NuMa, p53, PAK-2, PARP, Perforin, PITSLRE, PKC-delta, pRb,Presenilin, prICE, RAIDD, Ras, RIP, Sphingomyelinase, SREBPs, thymidinekinase from Herpes simplex, TNF-α, TNF-α receptor, TRADD, TRAF2,TRAIL-R1, TRAIL-R2, TRAIL-R3, Transglutaminase, U1 70 kDa snRNP, YAMA,etc.

In some embodiments, the heterologous gene encodes an antibody, or aportion, fragment, or variant thereof. Antibodies include fragments thatare capable of binding to an antigen, such as Fv, single-chain Fv(scFv), Fab, Fab′, di-scFv, sdAb (single domain antibody) and (Fab′)₂(including a chemically linked F(ab′)₂). Papain digestion of antibodiesproduces two identical antigen-binding fragments, called “Fab”fragments, each with a single antigen-binding site, and a residual “Fc”fragment, whose name reflects its ability to crystallize readily. Pepsintreatment yields an F(ab′)₂ fragment that has two antigen-combiningsites and is still capable of cross-linking antigen. Antibodies alsoinclude chimeric antibodies and humanized antibodies. Furthermore, forall antibody constructs provided herein, variants having the sequencesfrom other organisms are also contemplated. Thus, if a human version ofan antibody is disclosed, one of skill in the art will appreciate how totransform the human sequence based antibody into a mouse, rat, cat, dog,horse, etc. sequence. Antibody fragments also include either orientationof single chain scFvs, tandem di-scFv, diabodies, tandem tri-sdcFv,minibodies, etc. In some embodiments, such as when an antibody is anscFv, a single polynucleotide of a heterologous gene encodes a singlepolypeptide comprising both a heavy chain and a light chain linkedtogether. Antibody fragments also include nanobodies (e.g., sdAb, anantibody having a single, monomeric domain, such as a pair of variabledomains of heavy chains, without a light chain). Multispecificantibodies (e.g., bispecific antibodies, trispecific antibodies, etc.)are known in the art and contemplated as expression products of theheterologous genes of the present invention.

In some embodiments, the heterologous gene encodes all or a portion of atumor suppressor gene (e.g., a gene encoding intracellular proteins thatcontrol progression into a specific stage of the cell cycle (for e.g.,RB1); a gene encoding a receptor or signal transducer for a secretedhormone or developmental signal that inhibits cell proliferation (e.g.,adenomatous polyposis coli (APC)); a gene encoding a checkpoint-controlprotein that trigger cell cycle arrest in response to DNA damage orchromosomal defects (e.g., breast cancer type 1 susceptibility protein(BRCA1), p16 (INK4), or p14 (ARF)); a gene encoding a protein thatinduces apoptosis (for e.g., PUMA, NOXA, and BIM); or a gene encoding aprotein involved in repairing mistakes in DNA (for e.g., DNA mismatchrepair protein 2 (MSH2)).

In some embodiments, the heterologous gene encodes all or a portion of aloss of function of a transcription factor (e.g., a master regulator,e.g., a master regulator associate with a disease). In some embodiments,the heterologous gene is TSHZ2, HOXA2, MEIS2, HOXA3, HAND2, HOXA5,TBX18, PEG3, GLI2, CLOCK, HNF4A, VHL/HIF, WT-1, GSK-3, SPINT2, SMAD2,SMAD3, or SMAD4. In some embodiments, the heterologous gene encodes allor a portion of an epigenetic regulator. In some embodiments, theepigenetic regulator is a histone methyltransferase, such as SETDB1,PRMT5, etc. In some embodiments, the epigenetic regulator is a histonedemethylase, such as a histone lysine demethylase (KDM), etc. In someembodiments, the epigenetic regulator is a histone acetylase (HDAC). Insome embodiments, the epigenetic regulator is a DNA methyltransferase(DNMT). In some embodiments, the epigenetic regulator is a DNAdemethylase (e.g., TET1-3).

In some embodiments, the heterologous gene includes a reporter sequence,which can be useful in verifying heterologous gene expression, forexample, in specific cells and tissues. Reporter sequences that may beprovided in a transgene include, without limitation, DNA sequencesencoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase,thymidine kinase, green fluorescent protein (GFP), chloramphenicolacetyltransferase (CAT), luciferase, and others well known in the art.When associated with regulatory elements which drive their expression,the reporter sequences provide signals detectable by conventional means,including enzymatic, radiographic, colorimetric, fluorescence or otherspectrographic assays, fluorescent activating cell sorting assays andimmunological assays, including enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example,where the marker sequence is the LacZ gene, the presence of the vectorcarrying the signal is detected by assays for β-galactosidase activity.Where the transgene is green fluorescent protein or luciferase, thevector carrying the signal may be measured visually by color or lightproduction in a luminometer.

In some embodiments, the heterologous gene does not include a codingsequence. Non-coding sequences such as shRNA, promoters, enhancers,sequences to mark DNA (e.g., for antibody recognition), PCRamplification sites, sequences that define restriction enzyme sites,site-specific recombinase recognition sites, sequences that arerecognized by a protein that binds to and/or modifies nucleic acids, andlinkers, may be included in the vector. In instances in which aheterologous gene is a trans-splicing molecule, non-coding sequencesinclude binding domains that bind a target intron. In some embodiments,the heterologous gene includes a binding domain (e.g., a binding domain,e.g., a pre-mRNA binding portion of a trans-splicing molecule).

In some embodiments, the heterologous gene is from 0.1 Kb to 100 Kb inlength (e.g., the heterologous gene is from 0.2 Kb to 90 Kb, from 0.5 Kbto 80 Kb, from 1.0 Kb to 70 Kb, from 1.5 Kb to 60 Kb, from 2.0 Kb to 50Kb, from 2.5 Kb to 45 Kb, from 3.0 Kb to 40 Kb, from 3.5 Kb to 35 Kb,from 4.0 Kb to 30 Kb, from 4.5 Kb to 25 Kb, from 4.6 Kb to 24 Kb, from4.7 Kb to 23 Kb, from 4.8 Kb to 22 Kb, from 4.9 Kb to 21 Kb, from 5.0 Kbto 20 Kb, from 5.5 Kb to 18 Kb, from 6.0 Kb to 17 Kb, from 6.5 Kb to 16Kb, from 7.0 Kb to 15 Kb, from 7.5 Kb to 14 Kb, from 8.0 Kb to 13 Kb,from 8.5 Kb to 12.5 Kb, from 9.0 Kb to 12.0 Kb, from 9.5 Kb to 11.5 Kb,or from 10.0 Kb to 11.0 Kb in length, e.g., from 0.1 Kb to 0.5 Kb, from0.5 Kb to 1.0 Kb, from 1.0 Kb to 2.5 Kb, from 2.5 Kb to 4.5 Kb, from 4.5Kb to 8 Kb, from 8 Kb to 10 Kb, from 10 Kb to 15 Kb, from 15 Kb to 20 Kbin length, or greater, e.g., from 0.1 Kb to 0.25 Kb, from 0.25 Kb to 0.5Kb, from 0.5 Kb to 1.0 Kb, from 1.0 Kb to 1.5 Kb, from 1.5 Kb to 2.0 Kb,from 2.0 Kb to 2.5 Kb, from 2.5 Kb to 3.0 Kb, from 3.0 Kb to 3.5 Kb,from 3.5 Kb to 4.0 Kb, from 4.0 Kb to 4.5 Kb, from 4.5 Kb to 5.0 Kb,from 5.0 Kb to 5.5 Kb, from 5.5 Kb to 6.0 Kb, from 6.0 Kb to 6.5 Kb,from 6.5 Kb to 7.0 Kb, from 7.0 Kb to 7.5 Kb, from 7.5 Kb to 8.0 Kb,from 8.0 Kb to 8.5 Kb, from 8.5 Kb to 9.0 Kb, from 9.0 Kb to 9.5 Kb,from 9.5 Kb to 10 Kb, from 10 Kb to 10.5 Kb, from 10.5 Kb to 11 Kb, from11 Kb to 11.5 Kb, from 11.5 Kb to 12 Kb, from 12 Kb to 12.5 Kb, from12.5 Kb to 13 Kb, from 13 Kb to 13.5 Kb, from 13.5 Kb to 14 Kb, from 14Kb to 14.5 Kb, from 14.5 Kb to 15 Kb, from 15 Kb to 15.5 Kb, from 15.5Kb to 16 Kb, from 16 Kb to 16.5 Kb, from 16.5 Kb to 17 Kb, from 17 Kb to17.5 Kb, from 17.5 Kb to 18 Kb, from 18 Kb to 18.5 Kb, from 18.5 Kb to19 Kb, from 19 Kb to 19.5 Kb, from 19.5 Kb to 20 Kb, from 20 Kb to 21Kb, from 21 Kb to 22 Kb, from 22 Kb to 23 Kb, from 23 Kb to 24 Kb, from24 Kb to 25 Kb in length, or greater, e.g., about 4.5 Kb, about 5.0 Kb,about 5.5 Kb, about 6.0 Kb, about 6.5 Kb, about 7.0 Kb, about 7.5 Kb,about 8.0 Kb, about 8.5 Kb, about 9.0 Kb, about 9.5 Kb, about 10 Kb,about 11 Kb, about 12 Kb, about 13 Kb, about 14 Kb, about 15 Kb, about16 Kb, about 17 Kb, about 18 Kb, about 19 Kb, about 20 Kb in length, orgreater).

In some embodiments, the heterologous gene is at least 1,100 bp inlength (e.g., from 1,100 bp to 10,000 bp, from 1,100 bp to 8,000 bp, orfrom 1,100 bp to 5,000 bp in length).

In some embodiments, the heterologous gene is at least 2,500 bp inlength (e.g., from 2,500 bp to 10,000 bp, from 2,500 bp to 8,000 bp, orfrom 2,500 bp to 5,000 bp in length). For example, in particularembodiments, the heterologous gene is sufficiently large to encode aprotein and is not an oligonucleotide therapy (e.g., not an antisense,siRNA, shRNA therapy etc.).

Control Elements

In addition to the terminal repeat sequence (e.g., a DD element) and theheterologous gene, DNA vectors of the invention (e.g., circular DNAvectors as described herein) may include conventional control elementsnecessary which are operably linked to the heterologous gene in a mannerwhich permits transcription, translation, and/or expression in a targetcell.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter, and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and sequences that enhance secretion of theencoded product. Various expression control sequences, includingpromoters which are native, constitutive, inducible, and/ortissue-specific, are known in the art and may be utilized as part of thepresent invention. A promoter region is operably linked to aheterologous gene if the promoter region is capable of effectingtranscription of that gene such that the resulting transcript might betranslated into the desired protein or polypeptide. Promoters useful aspart of the DNA vectors described herein include constitutive andinducible promoters. Examples of constitutive promoters include, acytomegalovirus (CMV) promoter (optionally with the CMV enhancer), aretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), an SV40 promoter, a dihydrofolate reductase promoter, aβ-actin promoter, a phosphoglycerol kinase (PGK) promoter, and an EF1 apromoter.

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources. Many other systems havebeen described and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include zinc-inducible sheep metallothionine (MT) promoters,dexamethasone-inducible mouse mammary tumor virus promoters, T7polymerase promoter systems, ecdysone insect promoters,tetracycline-repressible systems, tetracycline-inducible systems,RU486-inducible systems, and rapamycin-inducible systems. Still othertypes of inducible promoters which may be useful in this context arethose which are regulated by a specific physiological state, e.g.,temperature, acute phase, a particular differentiation state of thecell, or in replicating cells only.

In another embodiment, the native promoter for the heterologous gene isused. The native promoter may be preferred when it is desired thatexpression of the heterologous gene should mimic the native expression.The native promoter may be used when expression of the heterologous genemust be regulated temporally or developmentally, or in a tissue-specificmanner, or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites, or Kozak consensus sequences may alsobe used to mimic native expression.

For heterologous genes encoding proteins, a polyadenylation (pA)sequence can be inserted following the heterologous gene and before theterminal repeat sequence. A heterologous gene useful in the presentdisclosure may also contain an intron, desirably located between thepromoter/enhancer sequence and the heterologous gene. Selection ofintrons and other common vector elements are conventional and many suchsequences are available.

The precise nature of the regulatory sequences needed for geneexpression in host cells may vary between species, tissues or celltypes, but shall in general include, as necessary, 5′ non-transcribedand 5′ non-translated sequences involved with the initiation oftranscription and translation respectively, such as a TATA box, cappingsequence, CAAT sequence, enhancer elements, and the like. Especially,such 5′ non-transcribed regulatory sequences will include a promoterregion that includes a promoter sequence for transcriptional control ofthe operably joined gene. Regulatory sequences may also include enhancersequences or upstream activator sequences as desired. The vectors of thedisclosure may optionally include 5′ leader or signal sequences.

III. Methods of Production

Provided herein are methods of producing a synthetic DNA vector (e.g., acircular DNA vector as described herein and/or a DNA vector having a DDelement). In particular, the methods provided herein involve in vitrosynthesis (e.g., in the absence of cells (i.e., cell-free)) rather bybacterial cell synthesis, which provides a purer composition ofresulting DNA vector relative to bacterial-derived vector and enables afaster, more efficient synthesis.

Cell-free synthesis of DNA vectors (e.g., circular DNA vectors asdescribed herein and/or DNA vectors containing a DD element) relies oneffective replication using a polymerase, such as a phage polymerase(e.g., Phi29 polymerase). In some embodiments, Phi29 polymerase isparticular useful to process replication of terminal repeat sequences,such as DD elements. The polymerase used herein can be a thermophilicpolymerase that has high processivity through GC-rich residues. In someembodiments, the polymerase used to replicate (e.g., amplify) the DDelement is Phi29 polymerase. Particular methods of producing the DNAvectors of the invention are described in detail in the Examples, below.

In general, production of a DNA vector (e.g., a circular DNA vector asdescribed herein) of the invention can begin with providing a samplehaving a circular DNA molecule including an AAV genome (e.g., a rAAVgenome) having heterologous gene and a terminal repeat sequence (e.g., aDD element). For example, the sample can be a lysate or otherpreparation from a cell (e.g., a mammalian cell) that was infected withthe AAV vector (e.g., rAAV vector). Double stranded circular DNA can beobtained from the cell using standard DNA extraction/isolationtechniques. In some embodiments, linear DNA is specifically degraded,e.g., using plasmid-safe DNase, to purify the circular DNA.

Next, the double stranded circular DNA having the AAV genome can beamplified in vitro, in a cell-free preparation, by incubating the DNAwith a polymerase (e.g., a phage polymerase, e.g., Phi29 DNA polymerase;TempliPhi kit, GE Healthcare), primers (e.g., random primers, e.g.,random hexamer primers), and a nucleotide mixture (e.g., dNTP, e.g.,dATP, dCTP, dGTP, and dTTP). In some embodiments, the nucleotide mixtureis a natural nucleotide mixture (i.e., substantially devoid ofnucleotide analogues). The polymerase (e.g., phage polymerase, e.g.,Phi29 polymerase) amplifies the AAV genome (e.g., an AAV genomeincluding an intact terminal repeat sequence, e.g., a DD element)) byrolling-circle amplification (e.g., isothermal rolling-circleamplification), generating a linear concatamer having a plurality of AAVgenome copies. Suitable polymerases include thermophilic polymerases andpolymerases that feature high processivity through GC-rich residues.

The resulting concatamers can be digested using a restriction enzyme tocut once within the genome to generate unit-length linear AAV genomesincluding the heterologous gene and the terminal repeat sequence (e.g.,a DD element)). Self-ligation of this linear DNA molecule (e.g., by theaddition of a DNA ligase) results in a circular, synthetic DNA vector ofthe invention, complete with the heterologous gene and, optionally, theintact terminal repeat sequence (e.g., a DD element). Alternatively,prior to self-ligation, the linear DNA molecule can be cloned into aplasmid vector according to known techniques and characterized, as isillustrated in the Examples below, prior to self-ligation to form thefinal DNA vector (e.g., a circular vector as described herein and/or aDD-containing DNA vector).

Because the replication and amplification of the genome is feasibleusing a polymerase in cell-free conditions, the synthetic DNA vector canbe isolated from the bacterial components of a plasmid in which it wascloned, and bacterial signatures, such as bacterial CpG motifs and/ordam or dcm methylation, are absent from the isolated vector.

IV. Pharmaceutical Compositions

Provided herein are pharmaceutical compositions including any of the DNAvectors (e.g., synthetic DNA vectors) described herein (e.g., DNAvectors containing a DD element and/or circular DNA vectors describedabove) in a pharmaceutically acceptable carrier. The pharmaceuticalcompositions described herein are substantially devoid of contaminates,such viral particles, viral capsid proteins, or peptide fragmentsthereof. In some embodiments, the pharmaceutical compositions providedherein are non-immunogenic. For example, non-immunogenic pharmaceuticalcompositions may be substantially devoid of pathogen-associatedmolecular patterns recognizable by cells of the innate immune system.Such pathogen-associated molecular patterns include CpG motifs (e.g.,unmethylated CpG motifs or hypomethylated CpG motifs), endotoxins (e.g.,lipopolysaccharides (LPS), e.g., bacterial LPS), flagellin, lipoteichoicacid, peptidoglycan, and viral nucleic acids molecules, such asdouble-stranded RNA.

The pharmaceutical compositions described herein may be assessed forcontamination by conventional methods and formulated into apharmaceutical composition intended for a suitable route ofadministration. Still other compositions containing the DNA vector maybe formulated similarly with a suitable carrier. Such formulationinvolves the use of a pharmaceutically and/or physiologically acceptablevehicle or carrier, particularly directed for administration to thetarget cell. In one embodiment, carriers suitable for administration tothe target cells include buffered saline, an isotonic sodium chloridesolution, or other buffers, e.g., HEPES, to maintain pH at appropriatephysiological levels, and, optionally, other medicinal agents,pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants,or diluents.

In some embodiments, the carrier is a liquid for injection. Exemplaryphysiologically acceptable carriers include sterile, pyrogen-free waterand sterile, pyrogen-free, phosphate buffered saline. A variety of suchknown carriers are provided in U.S. Pat. No. 7,629,322, incorporatedherein by reference. In one embodiment, the carrier is an isotonicsodium chloride solution. In another embodiment, the carrier is balancedsalt solution. In one embodiment, the carrier includes tween. If thevector is to be stored long-term, it may be frozen in the presence ofglycerol or Tween20.

In other embodiments, compositions containing vectors described hereininclude a surfactant. Useful surfactants, such as Pluronic F68(Poloxamer 188, also known as LUTROL® F68) may be included as theyprevent AAV from sticking to inert surfaces and thus ensure delivery ofthe desired dose. The carrier is isotonic sodium chloride solution andincludes a surfactant Pluronic F68.

Delivery vehicles such as liposomes, nanoparticles, microparticles,microspheres, lipid particles, vesicles, and the like, may be used forthe introduction of the compositions of the present disclosure intosuitable host cells. In particular, the DNA vectors may be formulatedfor delivery by encapsulation in a lipid particle, a liposome, avesicle, or a nanoparticle. In some embodiments, the DNA vector iscomplexed with a delivery vehicle such as a poloxamer and/orpolycationic material.

Pharmaceutical compositions having any of the DNA vectors of theinvention (e.g., circular DNA vectors as described herein and/or DNAvectors including a DD element) may contain a unit dose containing aquantity of DNA from 10 μg to 10 mg (e.g., from 25 μg to 5.0 mg, from 50μg to 2.0 mg, or from 100 μg to 1.0 mg of DNA, e.g., from 10 μg to 20μg, from 20 μg to 30 μg, from 30 μg to 40 μg, from 40 μg to 50 μg, from50 μg to 75 μg, from 75 μg to 100 μg, from 100 μg to 200 μg, from 200 μgto 300 μg, from 300 μg to 400 μg, from 400 μg to 500 μg, from 500 μg to1.0 mg, from 1.0 mg to 5.0 mg, or from 5.0 mg to 10 mg of DNA, e.g.,about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 150μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400μg, about 450 μg, about 500 μg, about 600 μg, about 700 μg, about 750μg, about 1.0 mg, about 2.0 mg, about 2.5 mg, about 5.0 mg, about 7.5mg, or about 10 mg of DNA).

In some embodiments, pharmaceutical compositions contain at least about0.01% DNA vector by weight. For example, the pharmaceutical compositionsmay contain 0.01% to 80% DNA vector by weight (e.g., from 0.05% to 50%by weight, 0.1% to 10% by weight, 0.5% to 5% by weight, or 1% to 2.5% byweight of DNA vector, e.g., 0.01% to 0.05% by weight, 0.05% to 0.1% byweight, 0.1% to 0.5% by weight, 0.5% to 1.0% by weight, 1.0% to 2% byweight, 2% to 3% by weight, 3% to 5% by weight, 5% to 10% by weight, 10%to 20% by weight, or 20% to 50% by weight of DNA vector).

Pharmaceutical compositions of the invention can contain any of thesynthetic circular DNA vectors described herein in monomeric form (e.g.,greater than 50% monomeric, greater than 60% monomeric, greater than 70%monomeric, greater than 80% monomeric, greater than 90% monomeric,greater than 95% monomeric, greater than 97% monomeric, greater than 98%monomeric, or greater than 99% monomeric). In some embodiments, from 70%to 99.99% of the synthetic circular DNA vector molecules in thepharmaceutical composition are monomeric (e.g., from 70% to 99.9%, from70% to 99.5%, from 70% to 99%, from 75% to 99.9%, from 75% to 99.5%,from 75% to 99%, from 80% to 99.9%, from 80% to 99.5%, from 80% to 99%,from 85% to 99.9%, from 85% to 99.5%, from 85% to 99%, from 90% to99.9%, from 90% to 99.5%, from 90% to 99%, from 95% to 99.9%, from 95%to 99.5%, or from 95% to 99% of the synthetic circular DNA vectormolecules in the pharmaceutical composition are monomeric, e.g., about70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, or 99.9% of the synthetic circular DNA vector molecules in thepharmaceutical composition are monomeric).

V. Methods of Use

Provided herein are methods of inducing expression (e.g., episomalexpression) of a heterologous gene in a subject in need thereof (e.g.,as part of a gene therapy regimen) by administering to the subject anyof the DNA vectors (e.g., circular DNA vectors as described hereinand/or DNA vectors including a DD element) or pharmaceuticalcompositions thereof described herein. Cells of a subject that contain aheterologous gene can be characterized by examining the nucleic acidsequence (e.g., an RNA sequence, e.g., an mRNA sequence) of the hostcell, such as by Southern Blotting or PCR analysis, to assay for thepresence of the heterologous gene contained in the vector.Alternatively, the expression of the heterologous gene in the subjectcan be characterized (e.g., quantitatively or qualitatively) bymonitoring the progress of a disease associated with a defect ormutation in the target gene corresponding to the heterologous gene. Insome embodiments, the expression (e.g., episomal expression) of theheterologous gene is confirmed by observing a decline in one or moresymptoms associated with the disease.

Accordingly, the invention provides methods of treating a disease in asubject associated with a defect in a target gene (e.g., a genecorresponding to the heterologous gene) by administering to the subjectany of the DNA vectors (e.g., circular DNA vectors as described hereinand/or DNA vectors including a DD element) or pharmaceuticalcompositions thereof described herein. In some embodiments, the diseaseis an ocular disease. In some embodiments, the subject is being treatedfor Leber's congenital amaurosis (LCA, e.g., LCA 10) using a DNA vectorhaving a heterologous CEP290 gene or portion thereof (e.g., as part of atrans-splicing molecule). In some embodiments, the subject is beingtreated for Stargardt Disease using a DNA vector having a heterologousABCA4 gene or portion thereof (e.g., as part of a trans-splicingmolecule). In some embodiments, the subject is being treated forpseudoxanthoma elasticum using a DNA vector having a heterologous ABCC6gene or portion thereof (e.g., as part of a trans-splicing molecule). Insome embodiments, the subject is being treated for rod cone dystrophy(e.g., rod cone dystrophy 7) using a DNA vector having a heterologousRIMS1 gene or portion thereof (e.g., as part of a trans-splicingmolecule). In some embodiments, the subject is being treated forexudative vitreoretinopathy using a DNA vector having a heterologousLRP5 gene or portion thereof (e.g., as part of a trans-splicingmolecule). In some embodiments, the subject is being treated for JoubertSyndrome using a DNA vector having a heterologous CC2D2A gene or portionthereof (e.g., as part of a trans-splicing molecule). In someembodiments, the subject is being treated for CSNB-1C using a DNA vectorhaving a heterologous TRPM1 gene or portion thereof (e.g., as part of atrans-splicing molecule). In some embodiments, the subject is beingtreated for age-related macular degeneration using a DNA vector having aheterologous C3 gene or portion thereof (e.g., as part of atrans-splicing molecule). In some embodiments, the subject is beingtreated for retinitis pigmentosa 71 using a DNA vector having aheterologous IFT172 gene or portion thereof (e.g., as part of atrans-splicing molecule). In some embodiments, the subject is beingtreated for stickler syndrome (e.g., stickler syndrome 2) using a DNAvector having a heterologous COL11A1 gene or portion thereof (e.g., aspart of a trans-splicing molecule). In some embodiments, the subject isbeing treated for microcephaly and chorioretinopathy using a DNA vectorhaving a heterologous TUBGCP6 gene or portion thereof (e.g., as part ofa trans-splicing molecule). In some embodiments, the subject is beingtreated for retinitis pigmentosa (e.g., RP recessive) using a DNA vectorhaving a heterologous KIAA1549 gene or portion thereof (e.g., as part ofa trans-splicing molecule). In some embodiments, the subject is beingtreated for CSNB 2 using a DNA vector having a heterologous CACNA1F geneor portion thereof (e.g., as part of a trans-splicing molecule). In someembodiments, the subject is being treated for Usher syndrome (e.g.,Usher syndrome type 1B) using a DNA vector having a heterologous MYO7Agene or portion thereof (e.g., as part of a trans-splicing molecule). Insome embodiments, the subject is being treated for Wagner syndrome usinga DNA vector having a heterologous VCAN gene or portion thereof (e.g.,as part of a trans-splicing molecule). In some embodiments, the subjectis being treated for Usher syndrome type 2A using a DNA vector having aheterologous USH2A gene or portion thereof (e.g., as part of atrans-splicing molecule). In some embodiments, the subject is beingtreated for AMD 1 using a DNA vector having a heterologous HMCN1 gene orportion thereof (e.g., as part of a trans-splicing molecule).

The invention also provides methods of treating a disease or disorderselected from the group consisting of an ocular disorder, a liverdisorder, a neurological disorder, an immune disorder, a cancer, acardiovascular disorder, a blood coagulation disorder, a lysosomalstorage disorder, or type 2 diabetes. The disorder may be an oculardisorder that is a retinal dystrophy.

The disorder may be a Mendelian-heritable retinal dystrophy. The retinaldystrophy may be selected from the group consisting of LCA, StargardtDisease, pseudoxanthoma elasticum, rod cone dystrophy, exudativevitreoretinopathy, Joubert Syndrome, CSNB-1C, age-related maculardegeneration, retinitis pigmentosa, stickler syndrome, microcephaly andchorioretinopathy, retinitis pigmentosa, CSNB 2, Usher syndrome, andWagner syndrome.

The disorder may be a neurological disorder, wherein the neurologicaldisorder is a neurodegenerative disease. The neurodegenerative diseasemay be selected from the group consisting of Alzheimer's disease,Parkinson's disease, or multiple sclerosis. The neurodegenerativedisease may be an autoimmune disease of the central nervous system(CNS). The autoimmune disease of the CNS may be multiple sclerosis,encephalomyelitis, a paraneoplastic syndrome, autoimmune inner eardisease, or opsoclonus myoclonus syndrome. The neurological disorder maybe a cerebral infarction, spinal cord injury, central nervous systemdisorder, a neuropsychiatric disorder, or a channelopathy. Theneurological disorder may be a channelopathy selected from epilepsy or amigraine. The neurological disorder may be an anxiety disorder, a mooddisorder, a childhood disorder, a cognitive disorder, schizophrenia, asubstance related disorders, or an eating disorders. The neurologicaldisorder may be a symptom of a cerebral infarction, stroke, traumaticbrain injury, or spinal cord injury.

The lysosomal storage disorder may be selected from the group consistingof Tay-Sachs disease, Gaucher disease, Fabry disease, Pompe disease,Niemann-Pick disease, and mucopolysaccharidosis (MPS).

The cardiovascular disorder may be a degenerative heart disease, acoronary artery disease, an ischemia, angina pectoris, an acute coronarysyndrome, a peripheral vascular disease, a peripheral arterial disease,a cerebrovascular disease, or atherosclerosis. The cardiovasculardisorder may be a degenerative heart disease selected from the groupconsisting of an ischemic cardiomyopathy, a conduction disease, and acongenital defect.

The disorder may be an immune disorder which is an autoimmune disorder.The autoimmune disorder may be type 1 diabetes, multiple sclerosis,rheumatoid arthritis, lupus, encephalomyelitis, a paraneoplasticsyndrome, autoimmune inner ear disease, or opsoclonus myoclonussyndrome, autoimmune hepatitis, uveitis, autoimmune retinopathy,neuromyelitis optica, psoriatic arthritis, psoriasis, myasthenia gravis,chronic Lyme disease, celiac disease, chronic inflammatory demyelinatingpolyneuropathy, peripheral neuropathy, fibromyalgia, Hashimoto'sthyroiditis, ulcerative colitis, or Kawasaki disease.

The disease may be a liver disease selected from the group consisting ofhepatitis, Alagille syndrome, biliary atresia, liver cancer, cirrhosis,a cystic disease, Caroli's syndrome, congenital hepatic fibrosis, fattyliver, galactosemia, primary sclerosing cholangitis, tyrosinemia,glycogen storage disease, Wilson's disease, and an endocrine deficiency.The liver disease may be a liver cancer selected from the groupconsisting of a hepatocellular hyperplasia, a hepatocellular adenomas, afocal nodular hyperplasia, or a hepatocellular carcinoma.

The disease may be a cancer which is a blood cancer or a solid tissuecancer. The blood cancer may be acute lymphoblastic leukemia, acutemyeloblastic leukemia, chromic myelogenous leukemia, Hodgkin's disease,multiple myeloma, and non-Hodgkin's lymphoma. The solid tissue cancermay be a liver cancer, kidney cancer, a breast cancer, a prostatecancer, a gastric cancer, an esophageal cancer, a stomach cancer, anintestinal cancer, a colorectal cancer, a bladder cancer, a head andneck cancer, a skin cancer, or a brain cancer.

Any of the vectors of the present invention (e.g., circular DNA vectorsas described herein and/or DNA vectors containing a DD element) can beadministered to a subject in a dosage from 10 μg to 10 mg of DNA (e.g.,from 25 μg to 5.0 mg, from 50 μg to 2.0 mg, or from 100 μg to 1.0 mg ofDNA, e.g., from 10 μg to 20 μg, from 20 μg to 30 μg, from 30 μg to 40μg, from 40 μg to 50 μg, from 50 μg to 75 μg, from 75 μg to 100 μg, from100 μg to 200 μg, from 200 μg to 300 μg, from 300 μg to 400 μg, from 400μg to 500 μg, from 500 μg to 1.0 mg, from 1.0 mg to 5.0 mg, or from 5.0mg to 10 mg of DNA, e.g., about 10 μg, about 20 μg, about 30 μg, about40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg,about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg,about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 600 μg,about 700 μg, about 750 μg, about 1.0 mg, about 2.0 mg, about 2.5 mg,about 5.0 mg, about 7.5 mg, or about 10 mg of DNA).

In some embodiments, administration of a DNA vector of the invention(e.g., a circular DNA vector as described herein and/or a DNA vectorcontaining a DD element), or a composition thereof, is non-immunogenicor less likely to induce an immune response in a subject compared withadministration of other gene therapy vectors (e.g., plasmid DNA vectorsand viral vectors). Methods of assessing immunogenicity of a vector aredescribed above.

The synthetic DNA vectors provided herein (e.g., circular DNA vectors asdescribed herein and/or DNA vectors containing a DD element) can beamenable to repeat dosing due to their ability to infect target cellswithout triggering an immune response or inducing a reduced immuneresponse relative to an AAV vector, as discussed above. Thus, theinvention provides methods of repeatedly administering the vectors andpharmaceutical compositions described herein. Any of the aforementioneddosing quantities may be repeated at a suitable frequency and duration.In some embodiments, the subject receives a dose about twice per day,about once per day, about five times per week, about four times perweek, about three times per week, about twice per week, about once perweek, about twice per month, about once per month, about once every sixweeks, about once every two months, about once every three months, aboutonce every four months, twice per year, once yearly, or less frequently.In some embodiments, the number and frequency of doses corresponds withthe rate of turnover of the target cell. It will be understood that inlong-lived post-mitotic target cells transfected using the vectorsdescribed herein, a single dose of vector may be sufficient to maintainexpression of the heterologous gene within the target cell for asubstantial period of time. Thus, in other embodiments, a DNA vectorprovided herein may be administered to a subject in a single dose. Thenumber of occasions in which heterologous nucleic acid is delivered tothe subject can be that which is required to maintain a clinical (e.g.,therapeutic) benefit.

Methods of the invention include administration of a DNA vector (e.g., acircular DNA vector as described herein and/or a DNA vector containing aDD element) or pharmaceutical composition thereof through any suitableroute. The DNA vector or pharmaceutical composition thereof can beadministered systemically or locally, e.g., intravenously, ocularly(e.g., intravitreally, subretinally, by eye drop, intraocularly,intraorbitally), intramuscularly, intravitreally (e.g., by intravitrealinjection), intradermally, intrahepatically, intracerebrally,intramuscularly, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intrathecally, intranasally,intravaginally, intrarectally, intratumorally, subcutaneously,subconjunctivally, intravesicularly, mucosally, intrapericardially,intraumbilically, orally, topically, transdermally, by inhalation, byaerosolization, by injection (e.g., by jet injection), byelectroporation, by implantation, by infusion (e.g., by continuousinfusion), by localized perfusion bathing target cells directly, bycatheter, by lavage, in creams, or in lipid compositions.

Additionally, or alternatively, vectors can be administered to hostcells ex vivo, such as by cells explanted from an individual patient,followed by reimplantation of the host cells into a patient, e.g., afterselection for cells which have incorporated the vector. Thus, in someaspects, the disclosure provides transfected host cells and methods ofadministration thereof for treating a disease.

The invention provides methods of inducing episomal expression of aheterologous gene in a subject in need thereof. For example, episomalexpression of a heterologous gene encoding a therapeutic replacementprotein (e.g., a monogenic therapeutic replacement protein) can beinduced in a subject having a defect or absence of the protein to bereplaced (e.g., thereby causing a coagulation disorder, a lysosomalstorage disorder, or any other disorders treatable by proteinreplacement therapy) by administering to the subject the isolated DNAvector (or a composition thereof) of the invention (e.g., an isolatedcircular DNA vector that lacks an origin of replication and/or a drugresistance gene; a recombination site; and any other element associatedwith bacterial synthesis (e.g., dam and dcm methylation patterns)). Inparticular instances, episomal expression of a heterologous geneencoding Factor VII is induced in a subject having a Factor VIIdeficiency by administering to the subject a DNA vector of the inventionthat encodes Factor VII. In some instances, episomal expression of aheterologous gene encoding Factor VIII is induced in a subject havinghemophilia A by administering to the subject a DNA vector of theinvention that encodes Factor VIII. In other instances, episomalexpression of a heterologous gene encoding Factor IX is induced in asubject having hemophilia B by administering to the subject a DNA vectorof the invention that encodes Factor IX. In some instances, episomalexpression of a heterologous gene encoding Factor X is induced in asubject having a Factor X deficiency by administering to the subject aDNA vector of the invention that encodes Factor X. In other instances,episomal expression of a heterologous gene encoding Factor XI is inducedin a subject having a Factor XI deficiency by administering to thesubject a DNA vector of the invention that encodes Factor XI. In someinstances, episomal expression of a heterologous gene encoding FactorXIII is induced in a subject having a Factor XIII deficiency byadministering to the subject a DNA vector of the invention that encodesFactor XIII. In some instances, episomal expression of a heterologousgene encoding von Willebrand factor is induced in a subject having vonWillebrand disease by administering to the subject a DNA vector of theinvention that encodes von Willebrand factor. In other instances,episomal expression of a heterologous gene encoding protein C is inducedin a subject having a protein C deficiency by administering to thesubject a DNA vector of the invention that encodes protein C. In someinstances, episomal expression of a heterologous gene encodingantithrombin III is induced in a subject having an antithrombin IIIdeficiency by administering to the subject a DNA vector of the inventionthat encodes antithrombin III. In some instances, episomal expression ofa heterologous gene encoding fibrinogen is induced in a subject having afibrinogen deficiency by administering to the subject a DNA vector ofthe invention that encodes fibrinogen. In other instances, episomalexpression of a heterologous gene encoding C1-esterase inhibitor isinduced in a subject having hereditary angioedema by administering tothe subject a DNA vector of the invention that encodes C1-esteraseinhibitor. In some instances, episomal expression of a heterologous geneencoding alpha-1 protease inhibitor is induced in a subject having a1-P1deficiency by administering to the subject a DNA vector of the inventionthat encodes alpha-1 protease inhibitor. In some instances, episomalexpression of a heterologous gene encoding glucocerebrosidase is inducedin a subject having Gaucher disease by administering to the subject aDNA vector of the invention that encodes glucocerebrosidase. In otherinstances, episomal expression of a heterologous gene encodingalpha-L-iduronidase is induced in a subject having mucopolysaccharidosisI by administering to the subject a DNA vector of the invention thatencodes alpha-L-iduronidase. In some instances, episomal expression of aheterologous gene encoding iduronate sulfatase is induced in a subjecthaving mucopolysaccharidosis II by administering to the subject a DNAvector of the invention that encodes iduronate sulfatase. In someinstances, episomal expression of a heterologous gene encodingN-acetylgalactosamine-4-sulfatase is induced in a subject havingmucopolysaccharidosis VI by administering to the subject a DNA vector ofthe invention that encodes N-acetylgalactosamine-4-sulfatase. In otherinstances, episomal expression of a heterologous gene encodingN-acetylgalactosamine-6-sulfatase is induced in a subject havingmucopolysaccharidosis IVA by administering to the subject a DNA vectorof the invention that encodes N-acetylgalactosamine-6-sulfatase. In someinstances, episomal expression of a heterologous gene encoding heparansulfate sulfatase is induced in a subject having mucopolysaccharidosisIIIA by administering to the subject a DNA vector of the invention thatencodes heparan sulfate sulfatase. In some instances, episomalexpression of a heterologous gene encoding alpha-galactosidase A isinduced in a subject having Fabry disease by administering to thesubject a DNA vector of the invention that encodes alpha-galactosidaseA. In other instances, episomal expression of a heterologous geneencoding alpha-glucosidase is induced in a subject having Pompe diseaseby administering to the subject a DNA vector of the invention thatencodes alpha-glucosidase. In some instances, episomal expression of aheterologous gene encoding acid sphingomyelinase is induced in a subjecthaving Niemann-Pick type B disease by administering to the subject a DNAvector of the invention that encodes acid sphingomyelinase. In someinstances, episomal expression of a heterologous gene encodingarylsuphatase A is induced in a subject having metachromaticleukodystrophy by administering to the subject a DNA vector of theinvention that encodes arylsuphatase A. In other instances, episomalexpression of a heterologous gene encoding lysosomal acid lipase (LAL)is induced in a subject having LAL deficiency by administering to thesubject a DNA vector of the invention that encodes lysosomal acidlipase. In some instances, episomal expression of a heterologous geneencoding sucrase-isomaltase is induced in a subject havingsucraseisomaltase deficiency by administering to the subject a DNAvector of the invention that encodes sucrase-isomaltase. In someinstances, episomal expression of a heterologous gene encoding adenosimedeaminase (ADA) is induced in a subject having an ADA deficiency byadministering to the subject a DNA vector of the invention that encodesadenosime deaminase. In other instances, episomal expression of aheterologous gene encoding insulin-like growth factor 1 (IGF-1) isinduced in a subject having an IGF-1 deficiency (e.g., primary IGF-1deficiency) by administering to the subject a DNA vector of theinvention that encodes IGF-1. In some instances, episomal expression ofa heterologous gene encoding alkaline phosphatase is induced in asubject having hypophosphatasia by administering to the subject a DNAvector of the invention that encodes alkaline phosphatase. In someinstances, episomal expression of a heterologous gene encodingporphobilinogen deaminase is induced in a subject having acuteintermittent porphyria by administering to the subject a DNA vector ofthe invention that encodes porphobilinogen deaminase.

Additionally or alternatively, the present invention includes methods oftreating a subject having a disease or disorder by administering to thesubject the isolated DNA vector (or a composition thereof) of theinvention (e.g., an isolated circular DNA vector that lacks an origin ofreplication and/or a drug resistance gene; a recombination site; and anyother element associated with bacterial synthesis (e.g., dam and dcmmethylation patterns)). In particular instances, a subject having aFactor VII deficiency is treated by administering to the subject a DNAvector of the invention that encodes Factor VII. In some instances, asubject having hemophilia A is treated by administering to the subject aDNA vector of the invention that encodes Factor VIII. In otherinstances, a subject having hemophilia B is treated by administering tothe subject a DNA vector of the invention that encodes Factor IX. Insome instances, a subject having a Factor X deficiency is treated byadministering to the subject a DNA vector of the invention that encodesFactor X. In other instances, a subject having a Factor XI deficiency istreated by administering to the subject a DNA vector of the inventionthat encodes Factor XI. In some instances, a subject having a FactorXIII deficiency is treated by administering to the subject a DNA vectorof the invention that encodes Factor XIII. In some instances, a subjecthaving von Willebrand disease is treated by administering to the subjecta DNA vector of the invention that encodes von Willebrand factor. Inother instances, a subject having a protein C deficiency is treated byadministering to the subject a DNA vector of the invention that encodesprotein C. In some instances, a subject having an antithrombin IIIdeficiency is treated by administering to the subject a DNA vector ofthe invention that encodes antithrombin III. In some instances, asubject having a fibrinogen deficiency is treated by administering tothe subject a DNA vector of the invention that encodes fibrinogen. Inother instances, a subject having hereditary angioedema is treated byadministering to the subject a DNA vector of the invention that encodesC1-esterase inhibitor. In some instances, a subject having a1-P1deficiency is treated by administering to the subject a DNA vector ofthe invention that encodes alpha-1 protease inhibitor. In someinstances, a subject having Gaucher disease is treated by administeringto the subject a DNA vector of the invention that encodesglucocerebrosidase. In other instances, a subject havingmucopolysaccharidosis I is treated by administering to the subject a DNAvector of the invention that encodes alpha-L-iduronidase. In someinstances, a subject having mucopolysaccharidosis II is treated byadministering to the subject a DNA vector of the invention that encodesiduronate sulfatase. In some instances, a subject havingmucopolysaccharidosis VI is treated by administering to the subject aDNA vector of the invention that encodesN-acetylgalactosamine-4-sulfatase. In other instances, a subject havingmucopolysaccharidosis IVA is treated by administering to the subject aDNA vector of the invention that encodesN-acetylgalactosamine-6-sulfatase. In some instances, a subject havingmucopolysaccharidosis IIIA is treated by administering to the subject aDNA vector of the invention that encodes heparan sulfate sulfatase. Insome instances, a subject having Fabry disease is treated byadministering to the subject a DNA vector of the invention that encodesalpha-galactosidase A. In other instances, a subject having Pompedisease is treated by administering to the subject a DNA vector of theinvention that encodes alpha-glucosidase. In some instances, a subjecthaving Niemann-Pick type B disease is treated by administering to thesubject a DNA vector of the invention that encodes acidsphingomyelinase. In some instances, a subject having metachromaticleukodystrophy is treated by administering to the subject a DNA vectorof the invention that encodes arylsuphatase A. In other instances, asubject having LAL deficiency is treated by administering to the subjecta DNA vector of the invention that encodes lysosomal acid lipase. Insome instances, a subject having sucraseisomaltase deficiency is treatedby administering to the subject a DNA vector of the invention thatencodes sucrase-isomaltase. In some instances, a subject having an ADAdeficiency is treated by administering to the subject a DNA vector ofthe invention that encodes adenosime deaminase. In other instances, asubject having an IGF-1 deficiency (e.g., primary IGF-1 deficiency) istreated by administering to the subject a DNA vector of the inventionthat encodes IGF-1. In some instances, a subject having hypophosphatasiais treated by administering to the subject a DNA vector of the inventionthat encodes alkaline phosphatase. In some instances, a subject havingacute intermittent porphyria is treated by administering to the subjecta DNA vector of the invention that encodes porphobilinogen deaminase.

Assessment and Monitoring

Assessment of the efficiency of transfection of any of the vectorsdescribed herein can be performed using any method known in the art ordescribed herein. Isolating a transfected cell can also be performed inaccordance with standard techniques. For example, a cell comprising aheterologous gene can express a visible marker, such as a fluorescentprotein (e.g., GFP) or other reporter protein, encoded by the sequenceof the heterologous gene that aids in the identification and isolationof a cell or cells comprising the heterologous gene. A cell containing aheterologous gene can also express a selectable marker from the gene.Survival of the cell under certain conditions, for example exposure to acytotoxic substance or the lack of a nutrient or substrate ordinarilyrequired for survival, may be dependent on expression or lack ofexpression of a selectable marker. Thus, survival or lack of survival ofcells under such conditions allows for identification and isolationcells or colonies of cells that contain a heterologous gene. Cellscontaining a heterologous gene can also be characterized by examiningthe nucleic acid sequence (e.g., an RNA sequence, e.g., an mRNAsequence) of the host cell, such as by Southern Blotting or PCRanalysis, to assay for the presence of the heterologous gene containedin the vector.

Accordingly, methods of the present invention include, afteradministering any of the vectors described herein to a subject,subsequently detecting the expression of the heterologous gene in thesubject. Expression can be detected one week to four weeks afteradministration, one month to four months after administration, fourmonths to one year after administration, one year to five years afteradministration, or five years to twenty years after administration(e.g., at least one week, at least two weeks, at least one month, atleast four months, at least one year, at least two years, at least fiveyears, at least ten years after administration). At any of thesedetection timepoints, persistence (e.g., episomal persistence) of theDNA vector may be observed. In some embodiments, the persistence of thecircular DNA vector is from 5% to 50% greater, 50% to 100% greater,one-fold to five-fold, or five-fold to ten-fold (e.g., at least 5%, 10%,20%, 30%, 40%, 50%, 75%, one-fold, two-fold, three-fold, four-fold,five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, ormore) greater than a reference vector (e.g., a circular vector producedin bacteria or having one or more bacterial signatures not present inthe vector of the invention).

The examples that follow do not limit the scope of the embodimentsdescribed herein. One skilled in the art will appreciate thatmodifications can be made in the following examples which are intendedto be encompassed by the spirit and scope of the invention.

EXAMPLES

Recombinant AAV (rAAV) vectors have an established record ofhigh-efficiency gene transfer in a variety of model systems and are nowbeing tested as therapeutic modalities in a wide range of humandiseases. Studies in animals and humans have shown that rAAV vectorgenomes persist in vivo predominantly as circular episomes. The presentinvention is based on the discovery that such persistence can bereplicated using synthetic techniques to produce circular DNA vectors.Molecular analysis of rAAV episomal genomes isolated from both animalsand humans reveals that these circular genomes contain terminal repeatsequences. In some of the following examples, terminal repeat sequencesidentified within rAAV episomal genomes include a Double D (DD) element,which is a result of recombination of the inverted terminal repeats(ITRs) located at each end of the linear AAV genome, shown in FIG. 1 .Such synthetic DNA vectors can reduce immunogenicity and inflammation inthe host relative to vectors generated in bacteria, since DNA producedin bacteria contains inherent bacterial signatures (CpG motifs) as wellas impurities from the bacteria themselves (endotoxin, bacterial genomicDNA and RNA) that can lead to loss of the plasmid and gene expression invivo.

Example 1. Synthetic Production of DNA Vectors Having a DD Element

Step 1—Production of rAAV2-eGFP Virus, Followed by Cell Transduction.

Plasmid pAAV-BASIC-EGFP was obtained (Vector Biolabs, Malvern, Pa.),which contained AAV2 ITRs flanking an expression cassette consisting ofa CMV enhancer/promoter driving eGFP protein with a BGHpA signal. Theplasmid was used in a triple transfection strategy in HEK293T cells toproduce rAAV2-eGFP viral vectors. Two other plasmids used in the tripletransfection were AAV helper plasmids pRep-Cap2 (Part No. 0912; AppliedViromics, Fremont, Calif.) and pHELP (Part No. 0913; Applied Viromics,Fremont, Calif.). The cells were transfected using a calcium phosphatekit (Profection Mammalian Transfection System, Part No. TM012; Promega,Madison, Wis.). At 48 hours post-transfection, the cells were lysed byfreeze/thaw and treated with benzonase to generate a crude viral lysate.The virus titer in the crude lysate was determined to be 5.3×10¹²DNase-resistant particles (DRP)/mL by qPCR. To generate circular rAAVgenomes, HEK293T cells were infected with the rAAV2-eGFP virus with amultiplicity of infection (MOI) of 1×10⁵. FIG. 4 summarizes thisprocess.

Step 2—Cloning and Characterization of rAAV Genome with DD Element.

A summary of the cloning and characterization of rAAV genome having a DDelement is shown in FIG. 5 . Infected cells were harvested seven dayspost-infection and total cellular DNA was extracted from cells using aDNeasy Blood and Tissue kit (Qiagen; Germantown, Md.). To eliminateresidual linear rAAV genomes, the DNA was treated with plasmid-safeDNase (Lucigen, Middleton, Wis.), which specifically degrades linearDNA, leaving double-stranded circular rAAV genomes intact. Residualcircular rAAV genomes were amplified using a TEMPLIPHI™ kit (Part No.25640010, GE Healthcare; Pittsburgh, Pa.). The TEMPLIPHI™ kit containsPhi29 polymerase that uses isothermal rolling circle amplification (RCA)for the exponential amplification of circular DNA using bacteriophagePhi29 DNA polymerase. The result of Phi29 amplification is long linearconcatamers of DNA. This DNA is then digested with an enzyme (EcoRI)that cuts once within the rAAV genome to produce a unit-length genomethat is cloned into pBlueScript II KS+ plasmid (Part No. 212207, AgilentTechnologies; Chicago, Ill.).

The DD elements within the resulting clones were sequenced, and clone“TG-18,” was identified as having an intact DD element (no deletions orrearrangements) of 165 bp in length. The sequence of clone TG-18 isshown in FIG. 6A.

Step 3—Generation of Template for DD Vector Production

Having identified an rAAV genome that contained a DD element (cloneTG-18), the next step was to produce a circular template for downstreamproduction of the DD vector. Plasmid TG-18 was digested with therestriction enzyme EcoRI, which released the linear unit-length rAAVgenome from the plasmid backbone. The linear fragment was thenself-ligated (rather than being ligated with a heterologous piece ofDNA) to re-create a circular rAAV genome. Any linear fragments that werenot ligated to form a circular product were eliminated by plasmid-safeDNase treatment. An illustration of this process is shown in FIG. 7 .

Step 4—Production of DD Vector in a Test Tube

The circular rAAV genome produced in Step 3 originated in bacteria andcontains bacterial signatures that have the potential to reducepersistence and/or to be immunogenic in the host. Step 4 amplifies thiscircular template in a test tube to generate more rAAV genomes that aredevoid of bacterial signatures and contaminants. This is an advantageover traditional gene transfer vectors produced in bacteria. For testtube production, the circular template is amplified using a TEMPLIPHI™kit (Part #25640010, GE Healthcare, Pittsburgh, Pa.). The TEMPLIPHI™ kitcontains Phi29 polymerase that uses isothermal rolling circleamplification (RCA) for the exponential amplification of circular DNAusing bacteriophage Phi29 DNA polymerase. The result of Phi29amplification is long linear concatamers of DNA. We examined theamplified DNA to see if the DD element was faithfully replicated withPhi29 DNA polymerase. Results are shown in FIG. 8 .

The amplified DNA was first digested with SwaI, which cuts on eitherside of the DD element (FIG. 9 ) to release a fragment of 244 bp inlength. The SwaI fragment from the amplified DNA was the same size asthe SwaI fragment from the original TG-18 pBlueScript plasmid (FIG. 10 ,arrow), indicating that Phi29 can amplify the DD element. The integrityof the amplified DD element was further analyzed by digestion with AhdIthat cuts within the DD element. AhdI cuts once within the DD vector anddigests the concatameric DNA into 2.1 kb unit-length genomes, asdemonstrated in FIG. 11 (arrow).

Having demonstrated that the DD element within the DD vector can befaithfully amplified, the next step was to generate the final circularDD vector products. An outline of the production strategy is shown inFIGS. 12-14 . The circular rAAV genome produced in Step 3 is amplifiedusing Phi29 polymerase that uses isothermal RCA for the exponentialamplification of circular DNA using bacteriophage Phi29 DNA polymerase.The result of Phi29 amplification is long linear concatamers of DNA(FIG. 13A). This DNA is then digested with an enzyme (EcoRI) that cutsonce within the rAAV genome to produce an AAV genome (i.e., aunit-length AAV genome; FIG. 13A). This AAV genome is then self-ligatedto re-create a circular rAAV genome (FIG. 14A). Any linear fragmentsthat were not ligated to form a circular product was eliminated byplasmid-safe DNase treatment.

Step 5—Confirmation of Gene Expression of DD Vector

The last step in the in vitro production process is to confirm that theDD vector is biologically active (i.e., expresses the transgene incultured cells). DD-containing DNA vector containing the eGFP expressioncassette as a heterologous gene was transfected into HEK293T cells usingLipofectamine 2000 (Life Technologies, Carlsbad, Calif.). Cells wereanalyzed 48 hours later for GFP expression by immunofluorescence (FIGS.15A and 15B) or western blotting (FIG. 16 ).

Example 2. Synthetic Production of Circular DNA Vectors

Monomeric DNA vectors were produced in which the vectors contain nobacterial plasmid DNA sequences and are synthesized entirely in a testtube (no replication in bacteria required). Therefore, the synthetic DNAvectors can endow a given target cell with transgene DNA that behaveslike AAV viral DNA—without needing the virus itself. This strategyoffers several advantages over viral vectors. First, it allows deliveryof genes that are too large for packaging into common viral vectors.Furthermore, it enables repeat dosing, since there are no viral proteinsthat would trigger an immune response to prevent repeat dosing ofanother viral vector. In addition, the in vitro synthesis process has agreater potential for more efficient manufacturing relative to otherviral vectors.

An exemplary process for generating synthetic circular DNA vectors isshown in FIG. 17 . Amplification of a supercoiled monomeric DNA templatewas performed using phi29 polymerase to generate linear concatameric DNAhaving a restriction site that defines the boundaries between repeatedDNA fragments. The concatamers were digested using a restriction enzymethat cleaves the DNA into unit-length fragments. Next, DNA ligase wasadded to induce self-ligation of the DNA fragments, generating a mixtureof DNA structures including open relaxed circles and supercoiled DNAmonomers. This mixture was column purified using a thiophilic aromaticadsorption chromatography resin (Plasmidselect Xtra, GE Healthcare28-4024-01), which has a selectivity that allows supercoiled covalentlyclosed circular forms of plasmid DNA to be separated from open circularforms. Supercoiled DNA monomer obtained from this purification wasrecovered and can be used in the methods described herein or,alternatively, may serve as a template for additional amplification.

Example 3. Characterization of In-Vivo Persistence—GFP Expression

To characterize the degree of persistence of a synthetic circular DNAvector of the invention, mice are administered with three compositions,each including a different DNA vector: (1) plasmid CAG-GFP (SEQ ID NO:42) as a negative control of persistence; (2) ΔDD CAG-GFP (a syntheticcircular DNA vector lacking a DD element); and (3) DD CAG-GFP (asynthetic circular DNA vector having a DD element). Each group contains32 mice total (eight mice per time point), and each composition isadministered at 10 μg DNA per mouse by hydrodynamic injection. Eightmice from each group are sacrificed at each of the following timepoints: two weeks, four weeks, eight weeks, and sixteen weeks, and livertissue is harvested and processed at each time point. Expression of GFPin liver cells is quantified according to known methods and comparedacross groups at each time point. Synthetic circular CAG-GFP isdetermined to be highly persistent if liver cells from mice administeredwith synthetic circular CAG-GFP express higher levels of GFP incomparison to liver cells from mice administered with plasmid CAG-GFP.

Example 4. Characterization of In-Vivo Persistence—mSEAP Expression

Another study to characterize the degree of persistence of a syntheticcircular DNA vector of the invention involves heterologous expression ofmouse secreted alkaline phosphatase (mSEAP), which is not endogenouslyexpressed in mice. In this experiment, mice are administered with fourcompositions, each including a different DNA vector: (1) plasmidCAG-mSEAP as a negative control of persistence; (2) plasmidCAG-mSEAP-ΔCpG, which lacks CpG motifs; (3) ΔDD CAG-mSEAP-ΔCpG, whichlacks a DD element and CpG motifs; and (4) DD CAG-mSEAP ΔCpG, whichincludes a DD element and lacks CpG motifs. Each group contains 12 mice,and each composition is administered at 20 μg DNA per mouse byhydrodynamic injection. Two mice from each group are sacrificed at eachof the following time points: two weeks, four weeks, eight weeks, twelveweeks, sixteen weeks, and twenty-four weeks, and 200 μL blood iscollected. Serum concentration of mSEAP is quantified in each sampleaccording to known methods and compared across groups at each timepoint.

The effect of CpG motifs and/or a DD element on persistence isquantified by comparing mSEAP concentration across the experimentalgroups. For example, serum mSEAP levels are approximately equivalentacross experimental groups at early time points; however, miceadministered with vectors having higher persistence exhibit greaterconcentrations of mSEAP at later time points.

Numerated Embodiments

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

1. An isolated circular DNA vector comprising one or more heterologousgenes encoding a therapeutic replacement protein, wherein the DNA vectorlacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

2. The DNA vector of paragraph 1, wherein the DNA vector comprises aterminal repeat sequence.

3. The DNA vector of paragraph 2, wherein the terminal repeat sequenceis at least 10 bp in length.

4. The DNA vector of any one of paragraphs 1-3, wherein the terminalrepeat sequence is a DD element.

5. The DNA vector of any one of paragraphs 1-4, wherein the DNA vectorlacks an immunogenic bacterial signature.

6. The DNA vector of any one of paragraphs 1-5, wherein the DNA vectorlacks an RNA polymerase arrest site.

7. The DNA vector of any one of paragraphs 1-6, wherein the DNA vectoris substantially devoid of CpG islands.

8. The DNA vector of any one of paragraphs 1-7, wherein the therapeuticreplacement protein is secreted into blood.

9. The DNA vector of any one of paragraphs 1-8, wherein the one or moreheterologous genes comprises a trans-splicing molecule or a portionthereof (e.g., a binding domain).

10. The DNA vector of any one of paragraphs 1-9, wherein the DNA vectorcomprises one or more unmethylated GATC sequences, one or moreunmethylated CCAGG sequences, and/or one or more CCTGG sequences.

11. The DNA vector of any one of paragraphs 1-10, wherein theheterologous gene is greater than 4.5 Kb in length.

12. The DNA vector of any one of paragraphs 1-11, wherein the DNA vectoris double stranded.

13. The DNA vector of any one of paragraphs 1-12, wherein the DNA vectoris monomeric.

14. The DNA vector of any one of paragraphs 1-13, wherein the DNA vectoris supercoiled.

15. The DNA vector of any one of paragraphs 1-14, wherein thetherapeutic replacement protein is indicated for treatment of an oculardisorder.

16. The DNA vector of paragraph 15, wherein the ocular disorder is aretinal dystrophy.

17. The DNA vector of paragraph 16, wherein the retinal dystrophy isselected from the group consisting of leber's congenital amaurosis(LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy,exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C, age-relatedmacular degeneration, retinitis pigmentosa, stickler syndrome,microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB 2, Ushersyndrome, and Wagner syndrome.

18. The DNA vector of any one of paragraphs 1-14, wherein thetherapeutic replacement protein is indicated for treatment of a bloodcoagulation disorder.

19. The DNA vector of paragraph 18, wherein the blood coagulationdisorder is a hemophilia, von Willebrand's disease, factor XIdeficiency, a fibrinogen disorder, or a vitamin K deficiency.

20. The DNA vector of paragraph 19, wherein the coagulation disorder ischaracterized by a mutation in a gene encoding for fibrinogen,prothrombin, factor V, factor VII, factor VIII, factor X, factor XI,factor XIII, or an enzyme involved in posttranslational modificationsthereof, or an enzyme involved in vitamin K metabolism.

21. An isolated circular DNA vector comprising one or more heterologousgenes encoding an antigen-binding protein, wherein the DNA vector lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

22. The DNA vector of paragraph 21, wherein the antigen-binding proteinis an antibody or an antigen-binding fragment thereof.

23. The DNA vector of paragraph 21 or 22, wherein the antigen-bindingprotein binds TNF, LT, IFN-α, IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-21, EMAP-II, GM-CSF, EGF, HER2, HER3, FGF, PDGF, BDNF,CNTF, CSF, G-CSF, NGF, PEDF, TGF, VEGF, gonadotropin, insulin-likegrowth factor, CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40,CD45, CD69, CD80, CD86, CD90, PD-1, PD-L1, amyloid beta, alkalinephosphatase, amyloid protein A, CCR4, folate receptor, mucin 5AC,PCSK-9, phosphatidyl-serine, or sclerostin.

24. The DNA vector of paragraph 22, wherein the antigen-binding proteinis a monoclonal antibody, a bispecific antibody, or an antigen-bindingfragment.

25. An isolated circular DNA vector comprising one or more heterologousgenes encoding an enzyme, a growth factor, a hormone, an interleukin, aninterferon, a cytokine, an anti-apoptosis factor, an anti-diabeticfactor, a coagulation factor, an anti-tumor factor, a liver-secretedprotein, or a neuroprotective factor, wherein the DNA vector lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

26. The DNA vector of paragraph 25, wherein the growth factor is BDNF,CNTF, CSF, EGF, FGF, G-SCF, M-CSF, GM-CSF, NGF, PDGF, PEDF, TGF, VEGF,gonadotropin, or an insulin-like growth factor.

27. The DNA vector of paragraph 25, wherein the interleukin is IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, or IL-21.

28. The DNA vector of paragraph 25, wherein the interferon is IFN-α orIFN-γ.

29. The DNA vector of paragraph 25, wherein the coagulation factor isfactor V, factor VII, factor VIII, factor IX, factor X, factor XI,factor XII, factor XIII, or von Willebrand factor.

30. The DNA vector of paragraph 25, wherein the neuroprotective factoris selected from the group consisting of a neurotrophin, Kifap3, Bcl-xl,Crmp1, Chk.beta., CALM2, Caly, NPG11, NPT1, Eef1a1, Dhps, Cd151,Morf412, CTGF, LDH-A, Atl1, NPT2, Ehd3, Cox5b, Tuba1a, gamma-actin,Rpsa, NPG3, NPG4, NPG5, NPG6, NPG7, NPG8, NPG9, and NPG10.

31. The DNA vector of paragraph 30, wherein the neurotrophin is selectedfrom the group consisting of NGF, BDNF, NT-3, NT-4, and CNTF.

32. An isolated circular DNA vector comprising one or more heterologousgenes associated with a disorder selected from the group consisting ofan ocular disorder, a liver disorder, a neurological disorder, an immunedisorder, a cancer, a cardiovascular disorder, a blood coagulationdisorder, a lysosomal storage disorder, or type 2 diabetes, wherein theDNA vector lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

33. The DNA vector of paragraph 32, wherein the disorder is an oculardisorder that is a retinal dystrophy.

34. The DNA vector of paragraph 33, wherein the disorder is aMendelian-heritable retinal dystrophy.

35. The DNA vector of paragraph 33, wherein the retinal dystrophy isselected from the group consisting of leber's congenital amaurosis(LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy,exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C, age-relatedmacular degeneration, retinitis pigmentosa, stickler syndrome,microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB 2, Ushersyndrome, and Wagner syndrome.

36. The DNA vector of paragraph 32, wherein the coagulation disorder isa hemophilia, von Willebrand's disease, factor XI deficiency, afibrinogen disorder, or a vitamin K deficiency.

37. The DNA vector of paragraph 32, wherein the coagulation disorder ischaracterized by a mutation in a gene encoding for fibrinogen,prothrombin, factor V, factor VII, factor VIII, factor X, factor XI,factor XIII, or an enzyme involved in posttranslational modificationsthereof, or an enzyme involved in vitamin K metabolism.

38. The DNA vector of paragraph 32 or 37, wherein the coagulationdisorder is characterized by a mutation in FGA, FGB, FGG, F2, F5, F7,F10, F11, F13A, F13B, LMAN1, MCFD2, GGCX, or VKORC1.

39. The DNA vector of paragraph 32, wherein the neurological disorder isa neurodegenerative disease.

40. The DNA vector of paragraph 39, wherein the neurodegenerativedisease is selected from the group consisting of Alzheimer's disease,Parkinson's disease, and multiple sclerosis.

41. The DNA vector of paragraph 39, wherein the neurodegenerativedisease is an autoimmune disease of the central nervous system (CNS).

42. The DNA vector of paragraph 41, wherein the autoimmune disease ofthe CNS is multiple sclerosis, encephalomyelitis, a paraneoplasticsyndrome, autoimmune inner ear disease, or opsoclonus myoclonussyndrome.

43. The DNA vector of paragraph 32, wherein the neurological disorder isa cerebral infarction, spinal cord injury, central nervous systemdisorder, a neuropsychiatric disorder, or a channelopathy.

44. The DNA vector of paragraph 43, wherein the channelopathy isepilepsy or migraine.

45. The DNA vector of paragraph 32, wherein the neurological disorder isan anxiety disorder, a mood disorder, a childhood disorder, a cognitivedisorder, schizophrenia, a substance related disorder, or an eatingdisorder.

46. The DNA vector of paragraph 32, wherein the neurological disorder isa symptom of a cerebral infarction, stroke, traumatic brain injury, orspinal cord injury.

47. The DNA vector of paragraph 32, wherein the lysosomal storagedisorder is selected from the group consisting of Tay-Sachs disease,Gaucher disease, Fabry disease, Pompe disease, Niemann-Pick disease, andmucopolysaccharidosis (MPS).

48. The DNA vector of paragraph 32, wherein the cardiovascular disorderis a degenerative heart disease, a coronary artery disease, an ischemia,angina pectoris, an acute coronary syndrome, a peripheral vasculardisease, a peripheral arterial disease, a cerebrovascular disease, oratherosclerosis.

49. The DNA vector of paragraph 32, wherein the cardiovascular disorderis a degenerative heart disease selected from the group consisting of anischemic cardiomyopathy, a conduction disease, and a congenital defect.

50. The DNA vector of paragraph 32, wherein the immune disorder is anautoimmune disorder.

51. The DNA vector of paragraph 50, wherein the autoimmune disorder istype 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus,encephalomyelitis, a paraneoplastic syndrome, autoimmune inner eardisease, or opsoclonus myoclonus syndrome, autoimmune hepatitis,uveitis, autoimmune retinopathy, neuromyelitis optica, psoriaticarthritis, psoriasis, myasthenia gravis, chronic Lyme disease, celiacdisease, chronic inflammatory demyelinating polyneuropathy, peripheralneuropathy, fibromyalgia, Hashimoto's thyroiditis, ulcerative colitis,or Kawasaki disease.

52. The DNA vector of paragraph 32, wherein the disease is a liverdisease selected from the group consisting of hepatitis, Alagillesyndrome, biliary atresia, liver cancer, cirrhosis, a cystic disease,Caroli's syndrome, congenital hepatic fibrosis, fatty liver,galactosemia, primary sclerosing cholangitis, tyrosinemia, glycogenstorage disease, Wilson's disease, and an endocrine deficiency.

53. The DNA vector of paragraph 52, wherein the liver disease is a livercancer selected from the group consisting of a hepatocellularhyperplasia, a hepatocellular adenomas, a focal nodular hyperplasia, ora hepatocellular carcinoma.

54. The DNA vector of paragraph 33, wherein the cancer is a blood canceror a solid tissue cancer.

55. The DNA vector of paragraph 54, wherein the blood cancer is acutelymphoblastic leukemia, acute myeloblastic leukemia, chromic myelogenousleukemia, Hodgkin's disease, multiple myeloma, and non-Hodgkin'slymphoma.

56. The DNA vector of paragraph 54, wherein the solid tissue cancer is aliver cancer, kidney cancer, a breast cancer, a gastric cancer, anesophageal cancer, a stomach cancer, an intestinal cancer, a colorectalcancer, a bladder cancer, a head and neck cancer, a skin cancer, or abrain cancer.

57. The DNA vector of any one of paragraphs 1-56, wherein the disorderis a recessively inherited disorder.

58. The DNA vector of any one of paragraphs 1-57, wherein theheterologous gene is expressible in a target cell selected from thegroup consisting of a liver cell, a retinal cell, a stem cell, a neuralcell, a muscle cell, or a blood cell.

59. The DNA vector of any one of paragraphs 1-58, wherein theheterologous gene is expressible in a post-mitotic target cell.

60. The DNA vector of paragraph 58 or 59, wherein the target cell is aneural cell selected from the group consisting of a neuron, anastrocyte, an oligodendrocyte, and a Schwann cell.

61. The DNA vector of any one of paragraphs 1-60, wherein thetherapeutic protein is secreted into blood.

62. The DNA vector of any one of paragraphs 1-61, wherein the DNA vectorcomprises a promoter sequence upstream of the one or more heterologousgenes.

63. The DNA vector of any one of paragraphs 1-62, wherein the DNA vectorcomprises a polyadenylation site downstream of the one or moreheterologous genes.

64. The DNA vector of any one of paragraphs 1-63, wherein the one ormore heterologous genes comprises a trans-splicing molecule or a portionthereof (e.g., a binding domain).

65. The DNA vector of any one of paragraphs 1-64, wherein the DNA vectorcomprises a terminal repeat sequence.

66. An isolated circular DNA vector comprising one or more therapeuticnucleic acids, wherein the DNA vector:

-   -   (a) lacks an origin of replication and/or a drug resistance        gene;    -   (b) lacks a recombination site; and    -   (c) comprises a terminal repeat sequence.

67. The DNA vector of paragraph 66, wherein the therapeutic nucleic acidis an siRNA, shRNA, miRNA, or CRISPRi molecule.

68. The DNA vector of any one of paragraphs 65-67, wherein the terminalrepeat sequence is at least 10 bp in length.

69. The DNA vector of any one of paragraphs 65-68, wherein the terminalrepeat sequence is a DD element.

70. The DNA vector of any one of paragraphs 1-69, wherein the vectorcomprises a suicide gene.

71. The DNA vector of any one of paragraphs 1-70, wherein the DNA vectorlacks bacterial plasmid DNA.

72. The DNA vector of any one of paragraphs 1-66, wherein the DNA vectorcomprises one or more unmethylated GATC sequences, one or moreunmethylated CCAGG sequences, and/or one or more CCTGG sequences.

73. The DNA vector of any one of paragraph 1-72, wherein the DNA vector:

-   -   (a) lacks an immunogenic bacterial signature;    -   (b) lacks an RNA polymerase arrest site; and/or    -   (c) is substantially devoid of CpG islands.

74. The DNA vector of any one of paragraphs 1-73, wherein theheterologous gene is greater than 4.5 Kb in length.

75. The DNA vector of any one of paragraphs 1-74, wherein the DNA vectoris double stranded.

76. The DNA vector of any one of paragraphs 1-75, wherein the DNA vectoris monomeric.

77. The DNA vector of any one of paragraphs 1-76, wherein the DNA vectoris supercoiled.

78. A composition comprising a plurality of the DNA vectors of any oneof paragraphs 1-77.

79. The composition of paragraph 78, wherein at least 50% of theplurality of the DNA vectors comprises one or more unmethylated GATCsequences, one or more unmethylated CCAGG sequences, and/or one or moreCCTGG sequences.

80. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises a heterologous gene encoding a therapeutic protein, whereinthe DNA molecule lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

81. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises one or more heterologous genes encoding an antigen-bindingprotein, wherein the DNA molecule lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

82. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises one or more heterologous genes encoding an enzyme, a growthfactor, a hormone, an interleukin, an interferon, a cytokine, ananti-apoptosis factor, an anti-diabetic factor, a coagulation factor, ananti-tumor factor, a liver-secreted protein, or a neuroprotectivefactor, wherein the DNA molecule lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

83. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises one or more heterologous genes associated with a disorderselected from the group consisting of an ocular disorder, a liverdisorder, a neurological disorder, an immune disorder, a cancer, acardiovascular disorder, a blood coagulation disorder, a lysosomalstorage disorder, or type 2 diabetes, wherein the DNA molecule lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

84. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises one or more therapeutic nucleic acids, wherein the DNAmolecule:

-   -   (a) lacks an origin of replication and/or a drug resistance        gene;    -   (b) lacks a recombination site; and    -   (c) comprises a terminal repeat sequence.

85. The isolated linear DNA molecule of any one of paragraphs 80-84,comprising a restriction enzyme site.

86. The isolated linear DNA molecule of paragraph 85, wherein therestriction enzyme site is positioned between the heterologous gene anda terminal repeat sequence.

87. A method of producing the isolated DNA vector of any one ofparagraphs 1-77, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene;    -   (ii) amplifying the AAV genome using polymerase-mediated        rolling-circle amplification to generate a linear concatamer;    -   (iii) digesting the concatamer using a restriction enzyme to        generate multiple AAV genomes; and    -   (iv) allowing each of the multiple AAV genomes to self-ligate to        produce an isolated DNA vector comprising the heterologous gene.

88. The method of paragraph 87, wherein the AAV genome comprises aterminal repeat sequence.

89. The method of paragraph 87 or 88, further comprising columnpurifying the isolated DNA vector comprising the heterologous gene topurify supercoiled DNA from the isolated DNA vector.

90. A method of producing the isolated DNA vector of any one ofparagraphs 1-77, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene and a terminal repeat sequence;    -   (ii) amplifying the AAV genome using a first polymerase-mediated        rolling-circle amplification to generate a first linear        concatamer;    -   (iii) digesting the first linear concatamer using a restriction        enzyme to generate a first AAV genome;    -   (iv) cloning the first AAV genome into a plasmid vector;    -   (v) identifying a plasmid clone comprising a terminal repeat        sequence;    -   (vi) digesting the plasmid clone comprising the terminal repeat        sequence to generate a second AAV genome;    -   (vii) allowing the second AAV genome to self-ligate to produce a        circular DNA template;    -   (viii) amplifying the circular DNA template using second        polymerase-mediated rolling-circle amplification to generate a        second linear concatamer;    -   (ix) digesting the second linear concatamer using a restriction        enzyme to generate a third AAV genome; and    -   (x) allowing the third AAV genome to self-ligate to produce an        isolated DNA vector comprising the heterologous gene and the        terminal repeat sequence.

91. The method of any one of paragraphs 87-90, wherein thepolymerase-mediated rolling-circle amplification is isothermalrolling-circle amplification.

92. The method of any one of paragraphs 87-91, wherein the polymerase isPhi29 DNA polymerase.

93. A cell-free method of producing the isolated DNA vector of any oneof paragraphs 1-58, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene;    -   (ii) amplifying the AAV genome using polymerase-mediated        rolling-circle amplification to generate a linear concatamer;    -   (iii) digesting the concatamer using a restriction enzyme to        generate an AAV genome; and    -   (iv) allowing the AAV genome to self-ligate to produce the        isolated DNA vector comprising the heterologous gene.

94. The method of paragraph 93, further comprising column purifying theisolated DNA vector comprising the heterologous gene to purifysupercoiled DNA from the isolated DNA vector.

95. The method of paragraph 93 or 94, wherein the polymerase-mediatedrolling-circle amplification is isothermal rolling-circle amplification.

96. The method of any one of paragraphs 93-95, wherein the polymerase isPhi29 DNA polymerase.

97. A method of producing the isolated DNA vector of any one ofparagraphs 1-77, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene and a DD element;    -   (ii) amplifying the AAV genome using polymerase-mediated        rolling-circle amplification to generate a linear concatamer;    -   (iii) digesting the concatamer using a restriction enzyme to        generate multiple AAV genomes; and    -   (iv) allowing each of the multiple AAV genomes to self-ligate to        produce an isolated DNA vector comprising the heterologous gene        and the DD element.

98. A method of producing the isolated DNA vector of any one ofparagraphs 1-77, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene and a DD element;    -   (ii) amplifying the AAV genome using a first polymerase-mediated        rolling-circle amplification to generate a first linear        concatamer;    -   (iii) digesting the first linear concatamer using a restriction        enzyme to generate a first AAV genome;    -   (iv) cloning the first AAV genome into a plasmid vector;    -   (v) identifying a plasmid clone comprising a DD element;    -   (vi) digesting the plasmid clone comprising the DD element to        generate a second AAV genome;    -   (vii) allowing the second AAV genome to self-ligate to produce a        circular DNA template;    -   (viii) amplifying the circular DNA template using second        polymerase-mediated rolling-circle amplification to generate a        second linear concatamer;    -   (ix) digesting the second linear concatamer using a restriction        enzyme to generate a third AAV genome; and    -   (x) allowing the third AAV genome to self-ligate to produce an        isolated DNA vector comprising the heterologous gene and the DD        element.

99. The method of paragraph 97 or 98, wherein the polymerase-mediatedrolling-circle amplification is isothermal rolling-circle amplification.

100. The method of any one of paragraphs 97-99, wherein the polymeraseis Phi29 DNA polymerase.

101. A cell-free method of producing the isolated DNA vector of any oneof paragraphs 1-77, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene and a DD element;    -   (ii) amplifying the AAV genome using polymerase-mediated        rolling-circle amplification to generate a linear concatamer;    -   (iii) digesting the concatamer using a restriction enzyme to        generate an AAV genome; and    -   (iv) allowing the AAV genome to self-ligate to produce a        therapeutic DNA vector comprising the heterologous gene and the        DD element.

102. The method of paragraph 101, wherein the polymerase-mediatedrolling-circle amplification is isothermal rolling-circle amplification.

103. The method of paragraph 101 or 102, wherein the polymerase is Phi29DNA polymerase.

104. A method of inducing episomal expression of a heterologous gene ina subject in need thereof, the method comprising administering to thesubject the isolated DNA vector of any one of paragraphs 1-77 or thecomposition of paragraph 78 or 79.

105. A method of treating a disorder in a subject, the method comprisingadministering to the subject the isolated DNA vector of any one ofparagraphs 1-77 or the composition of paragraph 78 or 79 in atherapeutically effective amount.

106. The method of paragraph 104 or 105, wherein the isolated DNA vectoror the composition is administered repeatedly.

107. The method of any one of paragraphs 104-106, wherein the isolatedDNA vector or the composition is administered locally.

108. The method of paragraph 107, wherein the isolated DNA vector or thecomposition is administered intravitreally.

109. The method of any one of paragraphs 104-108, wherein the disorderis an ocular disorder.

110. The method of paragraph 109, wherein the ocular disorder is aMendelian-heritable retinal dystrophy.

111. The method of paragraph 110, wherein the ocular disorder is LCA,Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy,exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C, age-relatedmacular degeneration, retinitis pigmentosa, stickler syndrome,microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB 2, Ushersyndrome, or Wagner syndrome.

112. The method of any one of paragraphs 104-106, wherein the isolatedDNA vector or the composition is administered systemically.

113. The method of paragraph 112, wherein the disorder is a coagulationdisorder.

114. The method of paragraph 113, wherein the coagulation disorder ishemophilia, von Willebrand's disease, factor XI deficiency, a fibrinogendisorder, or a vitamin K deficiency.

Other embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

1. An isolated circular DNA vector comprising one or more heterologousgenes encoding a therapeutic replacement protein, wherein the DNA vectorlacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

2. The DNA vector of paragraph 1, wherein the DNA vector comprises aterminal repeat sequence.

3. The DNA vector of paragraph 2, wherein the terminal repeat sequenceis at least 10 bp in length.

4. The DNA vector of any one of paragraphs 1-3, wherein the terminalrepeat sequence is a DD element.

5. The DNA vector of any one of paragraphs 1-4, wherein the DNA vectorlacks an immunogenic bacterial signature.

6. The DNA vector of any one of paragraphs 1-5, wherein the DNA vectorlacks an RNA polymerase arrest site.

7. The DNA vector of any one of paragraphs 1-6, wherein the DNA vectoris substantially devoid of CpG islands.

8. The DNA vector of any one of paragraphs 1-7, wherein the therapeuticreplacement protein is secreted into blood.

9. The DNA vector of any one of paragraphs 1-8, wherein the one or moreheterologous genes comprises a trans-splicing molecule or a portionthereof (e.g., a binding domain).

10. The DNA vector of any one of paragraphs 1-9, wherein the DNA vectorcomprises one or more unmethylated GATC sequences, one or moreunmethylated CCAGG sequences, and/or one or more CCTGG sequences.

11. The DNA vector of any one of paragraphs 1-10, wherein theheterologous gene is greater than 4.5 Kb in length.

12. The DNA vector of any one of paragraphs 1-11, wherein the DNA vectoris double stranded.

13. The DNA vector of any one of paragraphs 1-12, wherein the DNA vectoris monomeric.

14. The DNA vector of any one of paragraphs 1-13, wherein the DNA vectoris supercoiled.

15. The DNA vector of any one of paragraphs 1-14, wherein thetherapeutic replacement protein is indicated for treatment of an oculardisorder.

16. The DNA vector of paragraph 15, wherein the ocular disorder is aretinal dystrophy.

17. The DNA vector of paragraph 16, wherein the retinal dystrophy isselected from the group consisting of leber's congenital amaurosis(LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy,exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C, age-relatedmacular degeneration, retinitis pigmentosa, stickler syndrome,microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB 2, Ushersyndrome, and Wagner syndrome.

18. The DNA vector of any one of paragraphs 1-14, wherein thetherapeutic replacement protein is indicated for treatment of a bloodcoagulation disorder.

19. The DNA vector of paragraph 18, wherein the blood coagulationdisorder is a hemophilia, von Willebrand's disease, factor XIdeficiency, a fibrinogen disorder, or a vitamin K deficiency.

20. The DNA vector of paragraph 18, wherein the coagulation disorder ischaracterized by a mutation in a gene encoding for fibrinogen,prothrombin, factor V, factor VII, factor VIII, factor X, factor XI,factor XIII, or an enzyme involved in posttranslational modificationsthereof, or an enzyme involved in vitamin K metabolism.

21. An isolated circular DNA vector comprising one or more heterologousgenes encoding an antigen-binding protein, wherein the DNA vector lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

22. The DNA vector of paragraph 21, wherein the antigen-binding proteinis an antibody or an antigen-binding fragment thereof.

23. The DNA vector of paragraph 21 or 22, wherein the antigen-bindingprotein binds TNF, LT, IFN-α, IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-21, EMAP-II, GM-CSF, EGF, HER2, HER3, FGF, PDGF, BDNF,CNTF, CSF, G-CSF, NGF, PEDF, TGF, VEGF, gonadotropin, insulin-likegrowth factor, CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40,CD45, CD69, CD80, CD86, CD90, PD-1, PD-L1, amyloid beta, alkalinephosphatase, amyloid protein A, CCR4, folate receptor, mucin 5AC,PCSK-9, phosphatidyl-serine, or sclerostin.

24. The DNA vector of paragraph 22, wherein the antigen-binding proteinis a monoclonal antibody, a bispecific antibody, or an antigen-bindingfragment.

25. An isolated circular DNA vector comprising one or more heterologousgenes encoding an enzyme, a growth factor, a hormone, an interleukin, aninterferon, a cytokine, an anti-apoptosis factor, an anti-diabeticfactor, a coagulation factor, an anti-tumor factor, a liver-secretedprotein, or a neuroprotective factor, wherein the DNA vector lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

26. The DNA vector of paragraph 25, wherein the growth factor is BDNF,CNTF, CSF, EGF, FGF, G-SCF, M-CSF, GM-CSF, NGF, PDGF, PEDF, TGF, VEGF,gonadotropin, or an insulin-like growth factor.

27. The DNA vector of paragraph 25, wherein the interleukin is IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, or IL-21.

28. The DNA vector of paragraph 25, wherein the interferon is IFN-α orIFN-γ.

29. The DNA vector of paragraph 25, wherein the coagulation factor isfactor V, factor VII, factor VIII, factor IX, factor X, factor XI,factor XII, factor XIII, or von Willebrand factor.

30. The DNA vector of paragraph 25, wherein the neuroprotective factoris selected from the group consisting of a neurotrophin, Kifap3, Bcl-xl,Crmp1, Chk.beta., CALM2, Caly, NPG11, NPT1, Eef1a1, Dhps, Cd151,Morf412, CTGF, LDH-A, Atl1, NPT2, Ehd3, Cox5b, Tuba1a, gamma-actin,Rpsa, NPG3, NPG4, NPG5, NPG6, NPG7, NPG8, NPG9, and NPG10.

31. The DNA vector of paragraph 30, wherein the neurotrophin is selectedfrom the group consisting of NGF, BDNF, NT-3, NT-4, and CNTF.

32. An isolated circular DNA vector comprising one or more heterologousgenes associated with a disorder selected from the group consisting ofan ocular disorder, a liver disorder, a neurological disorder, an immunedisorder, a cancer, a cardiovascular disorder, a blood coagulationdisorder, a lysosomal storage disorder, or type 2 diabetes, wherein theDNA vector lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

33. The DNA vector of paragraph 32, wherein the disorder is an oculardisorder that is a retinal dystrophy.

34. The DNA vector of paragraph 33, wherein the disorder is aMendelian-heritable retinal dystrophy.

35. The DNA vector of paragraph 33, wherein the retinal dystrophy isselected from the group consisting of leber's congenital amaurosis(LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy,exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C, age-relatedmacular degeneration, retinitis pigmentosa, stickler syndrome,microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB 2, Ushersyndrome, and Wagner syndrome.

36. The DNA vector of paragraph 32, wherein the coagulation disorder isa hemophilia, von Willebrand's disease, factor XI deficiency, afibrinogen disorder, or a vitamin K deficiency.

37. The DNA vector of paragraph 32, wherein the coagulation disorder ischaracterized by a mutation in a gene encoding for fibrinogen,prothrombin, factor V, factor VII, factor VIII, factor X, factor XI,factor XIII, or an enzyme involved in posttranslational modificationsthereof, or an enzyme involved in vitamin K metabolism.

38. The DNA vector of paragraph 32 or 37, wherein the coagulationdisorder is characterized by a mutation in FGA, FGB, FGG, F2, F5, F7,F10, F11, F13A, F13B, LMAN1, MCFD2, GGCX, or VKORC1.

39. The DNA vector of paragraph 32, wherein the neurological disorder isa neurodegenerative disease.

40. The DNA vector of paragraph 39, wherein the neurodegenerativedisease is selected from the group consisting of Alzheimer's disease,Parkinson's disease, and multiple sclerosis.

41. The DNA vector of paragraph 39, wherein the neurodegenerativedisease is an autoimmune disease of the central nervous system (CNS).

42. The DNA vector of paragraph 41, wherein the autoimmune disease ofthe CNS is multiple sclerosis, encephalomyelitis, a paraneoplasticsyndrome, autoimmune inner ear disease, or opsoclonus myoclonussyndrome.

43. The DNA vector of paragraph 32, wherein the neurological disorder isa cerebral infarction, spinal cord injury, central nervous systemdisorder, a neuropsychiatric disorder, or a channelopathy.

44. The DNA vector of paragraph 43, wherein the channelopathy isepilepsy or migraine.

45. The DNA vector of paragraph 32, wherein the neurological disorder isan anxiety disorder, a mood disorder, a childhood disorder, a cognitivedisorder, schizophrenia, a substance related disorder, or an eatingdisorder.

46. The DNA vector of paragraph 32, wherein the neurological disorder isa symptom of a cerebral infarction, stroke, traumatic brain injury, orspinal cord injury.

47. The DNA vector of paragraph 32, wherein the lysosomal storagedisorder is selected from the group consisting of Tay-Sachs disease,Gaucher disease, Fabry disease, Pompe disease, Niemann-Pick disease, andmucopolysaccharidosis (MPS).

48. The DNA vector of paragraph 32, wherein the cardiovascular disorderis a degenerative heart disease, a coronary artery disease, an ischemia,angina pectoris, an acute coronary syndrome, a peripheral vasculardisease, a peripheral arterial disease, a cerebrovascular disease, oratherosclerosis.

49. The DNA vector of paragraph 32, wherein the cardiovascular disorderis a degenerative heart disease selected from the group consisting of anischemic cardiomyopathy, a conduction disease, and a congenital defect.

50. The DNA vector of paragraph 32, wherein the immune disorder is anautoimmune disorder.

51. The DNA vector of paragraph 50, wherein the autoimmune disorder istype 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus,encephalomyelitis, a paraneoplastic syndrome, autoimmune inner eardisease, or opsoclonus myoclonus syndrome, autoimmune hepatitis,uveitis, autoimmune retinopathy, neuromyelitis optica, psoriaticarthritis, psoriasis, myasthenia gravis, chronic Lyme disease, celiacdisease, chronic inflammatory demyelinating polyneuropathy, peripheralneuropathy, fibromyalgia, Hashimoto's thyroiditis, ulcerative colitis,or Kawasaki disease.

52. The DNA vector of paragraph 32, wherein the disease is a liverdisease selected from the group consisting of hepatitis, Alagillesyndrome, biliary atresia, liver cancer, cirrhosis, a cystic disease,Caroli's syndrome, congenital hepatic fibrosis, fatty liver,galactosemia, primary sclerosing cholangitis, tyrosinemia, glycogenstorage disease, Wilson's disease, and an endocrine deficiency.

53. The DNA vector of paragraph 52, wherein the liver disease is a livercancer selected from the group consisting of a hepatocellularhyperplasia, a hepatocellular adenomas, a focal nodular hyperplasia, ora hepatocellular carcinoma.

54. The DNA vector of paragraph 33, wherein the cancer is a blood canceror a solid tissue cancer.

55. The DNA vector of paragraph 54, wherein the blood cancer is acutelymphoblastic leukemia, acute myeloblastic leukemia, chromic myelogenousleukemia, Hodgkin's disease, multiple myeloma, and non-Hodgkin'slymphoma.

56. The DNA vector of paragraph 54, wherein the solid tissue cancer is aliver cancer, kidney cancer, a breast cancer, a gastric cancer, anesophageal cancer, a stomach cancer, an intestinal cancer, a colorectalcancer, a bladder cancer, a head and neck cancer, a skin cancer, or abrain cancer.

57. The DNA vector of any one of paragraphs 1-56, wherein the disorderis a recessively inherited disorder.

58. The DNA vector of any one of paragraphs 1-57, wherein theheterologous gene is expressible in a target cell selected from thegroup consisting of a liver cell, a retinal cell, a stem cell, a neuralcell, a muscle cell, or a blood cell.

59. The DNA vector of any one of paragraphs 1-58, wherein theheterologous gene is expressible in a post-mitotic target cell.

60. The DNA vector of paragraph 58 or 59, wherein the target cell is aneural cell selected from the group consisting of a neuron, anastrocyte, an oligodendrocyte, and a Schwann cell.

61. The DNA vector of any one of paragraphs 1-60, wherein thetherapeutic protein is secreted into blood.

62. The DNA vector of any one of paragraphs 1-61, wherein the DNA vectorcomprises a promoter sequence upstream of the one or more heterologousgenes.

63. The DNA vector of any one of paragraphs 1-62, wherein the DNA vectorcomprises a polyadenylation site downstream of the one or moreheterologous genes.

64. The DNA vector of any one of paragraphs 1-63, wherein the one ormore heterologous genes comprises a trans-splicing molecule or a portionthereof (e.g., a binding domain).

65. The DNA vector of any one of paragraphs 1-64, wherein the DNA vectorcomprises a terminal repeat sequence.

66. An isolated circular DNA vector comprising one or more therapeuticnucleic acids, wherein the DNA vector:

-   -   (a) lacks an origin of replication and/or a drug resistance        gene;    -   (b) lacks a recombination site; and    -   (c) comprises a terminal repeat sequence.

67. The DNA vector of paragraph 66, wherein the therapeutic nucleic acidis an siRNA, shRNA, miRNA, or CRISPRi molecule.

68. The DNA vector of any one of paragraphs 65-67, wherein the terminalrepeat sequence is at least 10 bp in length.

69. The DNA vector of any one of paragraphs 65-68, wherein the terminalrepeat sequence is a DD element.

70. The DNA vector of any one of paragraphs 1-69, wherein the vectorcomprises a suicide gene.

71. The DNA vector of any one of paragraphs 1-70, wherein the DNA vectorlacks bacterial plasmid DNA.

72. The DNA vector of any one of paragraphs 1-66, wherein the DNA vectorcomprises one or more unmethylated GATC sequences, one or moreunmethylated CCAGG sequences, and/or one or more CCTGG sequences.

73. The DNA vector of any one of paragraph 1-72, wherein the DNA vector:

-   -   (a) lacks an immunogenic bacterial signature;    -   (b) lacks an RNA polymerase arrest site; and/or    -   (c) is substantially devoid of CpG islands.

74. The DNA vector of any one of paragraphs 1-73, wherein theheterologous gene is greater than 4.5 Kb in length.

75. The DNA vector of any one of paragraphs 1-74, wherein the DNA vectoris double stranded.

76. The DNA vector of any one of paragraphs 1-75, wherein the DNA vectoris monomeric.

77. The DNA vector of any one of paragraphs 1-76, wherein the DNA vectoris supercoiled.

78. A composition comprising a plurality of the DNA vectors of any oneof paragraphs 1-77.

79. The composition of paragraph 78, wherein at least 50% of theplurality of the DNA vectors comprises one or more unmethylated GATCsequences, one or more unmethylated CCAGG sequences, and/or one or moreCCTGG sequences.

80. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises a heterologous gene encoding a therapeutic protein, whereinthe DNA molecule lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

81. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises one or more heterologous genes encoding an antigen-bindingprotein, wherein the DNA molecule lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

82. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises one or more heterologous genes encoding an enzyme, a growthfactor, a hormone, an interleukin, an interferon, a cytokine, ananti-apoptosis factor, an anti-diabetic factor, a coagulation factor, ananti-tumor factor, a liver-secreted protein, or a neuroprotectivefactor, wherein the DNA molecule lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

83. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises one or more heterologous genes associated with a disorderselected from the group consisting of an ocular disorder, a liverdisorder, a neurological disorder, an immune disorder, a cancer, acardiovascular disorder, a blood coagulation disorder, a lysosomalstorage disorder, or type 2 diabetes, wherein the DNA molecule lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

84. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises one or more therapeutic nucleic acids, wherein the DNAmolecule:

-   -   (a) lacks an origin of replication and/or a drug resistance        gene;    -   (b) lacks a recombination site; and    -   (c) comprises a terminal repeat sequence.

85. The isolated linear DNA molecule of any one of paragraphs 80-84,comprising a restriction enzyme site.

86. The isolated linear DNA molecule of paragraph 85, wherein therestriction enzyme site is positioned between the heterologous gene anda terminal repeat sequence.

87. A method of producing the isolated DNA vector of any one ofparagraphs 1-77, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene;    -   (ii) amplifying the AAV genome using polymerase-mediated        rolling-circle amplification to generate a linear concatamer;    -   (iii) digesting the concatamer using a restriction enzyme to        generate multiple AAV genomes; and    -   (iv) allowing each of the multiple AAV genomes to self-ligate to        produce an isolated DNA vector comprising the heterologous gene.

88. The method of paragraph 87, wherein the AAV genome comprises aterminal repeat sequence.

89. The method of paragraph 87 or 88, further comprising columnpurifying the isolated DNA vector comprising the heterologous gene topurify supercoiled DNA from the isolated DNA vector.

90. A method of producing the isolated DNA vector of any one ofparagraphs 1-77, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene and a terminal repeat sequence;    -   (ii) amplifying the AAV genome using a first polymerase-mediated        rolling-circle amplification to generate a first linear        concatamer;    -   (iii) digesting the first linear concatamer using a restriction        enzyme to generate a first AAV genome;    -   (iv) cloning the first AAV genome into a plasmid vector;    -   (v) identifying a plasmid clone comprising a terminal repeat        sequence;    -   (vi) digesting the plasmid clone comprising the terminal repeat        sequence to generate a second AAV genome;    -   (vii) allowing the second AAV genome to self-ligate to produce a        circular DNA template;    -   (viii) amplifying the circular DNA template using second        polymerase-mediated rolling-circle amplification to generate a        second linear concatamer;    -   (ix) digesting the second linear concatamer using a restriction        enzyme to generate a third AAV genome; and    -   (x) allowing the third AAV genome to self-ligate to produce an        isolated DNA vector comprising the heterologous gene and the        terminal repeat sequence.

91. The method of any one of paragraphs 87-90, wherein thepolymerase-mediated rolling-circle amplification is isothermalrolling-circle amplification.

92. The method of any one of paragraphs 87-91, wherein the polymerase isPhi29 DNA polymerase.

93. A cell-free method of producing the isolated DNA vector of any oneof paragraphs 1-58, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene;    -   (ii) amplifying the AAV genome using polymerase-mediated        rolling-circle amplification to generate a linear concatamer;    -   (iii) digesting the concatamer using a restriction enzyme to        generate an AAV genome; and    -   (iv) allowing the AAV genome to self-ligate to produce the        isolated DNA vector comprising the heterologous gene.

94. The method of paragraph 93, further comprising column purifying theisolated DNA vector comprising the heterologous gene to purifysupercoiled DNA from the isolated DNA vector.

95. The method of paragraph 93 or 94, wherein the polymerase-mediatedrolling-circle amplification is isothermal rolling-circle amplification.

96. The method of any one of paragraphs 93-95, wherein the polymerase isPhi29 DNA polymerase.

97. A method of producing the isolated DNA vector of any one ofparagraphs 1-77, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene and a DD element;    -   (ii) amplifying the AAV genome using polymerase-mediated        rolling-circle amplification to generate a linear concatamer;    -   (iii) digesting the concatamer using a restriction enzyme to        generate multiple AAV genomes; and    -   (iv) allowing each of the multiple AAV genomes to self-ligate to        produce an isolated DNA vector comprising the heterologous gene        and the DD element.

98. A method of producing the isolated DNA vector of any one ofparagraphs 1-77, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene and a DD element;    -   (ii) amplifying the AAV genome using a first polymerase-mediated        rolling-circle amplification to generate a first linear        concatamer;    -   (iii) digesting the first linear concatamer using a restriction        enzyme to generate a first AAV genome;    -   (iv) cloning the first AAV genome into a plasmid vector;    -   (v) identifying a plasmid clone comprising a DD element;    -   (vi) digesting the plasmid clone comprising the DD element to        generate a second AAV genome;    -   (vii) allowing the second AAV genome to self-ligate to produce a        circular DNA template;    -   (viii) amplifying the circular DNA template using second        polymerase-mediated rolling-circle amplification to generate a        second linear concatamer;    -   (ix) digesting the second linear concatamer using a restriction        enzyme to generate a third AAV genome; and    -   (x) allowing the third AAV genome to self-ligate to produce an        isolated DNA vector comprising the heterologous gene and the DD        element.

99. The method of paragraph 97 or 98, wherein the polymerase-mediatedrolling-circle amplification is isothermal rolling-circle amplification.

100. The method of any one of paragraphs 97-99, wherein the polymeraseis Phi29 DNA polymerase.

101. A cell-free method of producing the isolated DNA vector of any oneof paragraphs 1-77, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene and a DD element;    -   (ii) amplifying the AAV genome using polymerase-mediated        rolling-circle amplification to generate a linear concatamer;    -   (iii) digesting the concatamer using a restriction enzyme to        generate an AAV genome; and    -   (iv) allowing the AAV genome to self-ligate to produce a        therapeutic DNA vector comprising the heterologous gene and the        DD element.

102. The method of paragraph 101, wherein the polymerase-mediatedrolling-circle amplification is isothermal rolling-circle amplification.

103. The method of paragraph 101 or 102, wherein the polymerase is Phi29DNA polymerase.

104. A method of inducing episomal expression of a heterologous gene ina subject in need thereof, the method comprising administering to thesubject the isolated DNA vector of any one of paragraphs 1-77 or thecomposition of paragraph 78 or 79.

105. A method of treating a disorder in a subject, the method comprisingadministering to the subject the isolated DNA vector of any one ofparagraphs 1-77 or the composition of paragraph 78 or 79 in atherapeutically effective amount.

106. The method of paragraph 104 or 105, wherein the isolated DNA vectoror the composition is administered repeatedly.

107. The method of any one of paragraphs 104-106, wherein the isolatedDNA vector or the composition is administered locally.

108. The method of paragraph 107, wherein the isolated DNA vector or thecomposition is administered intravitreally.

109. The method of any one of paragraphs 104-108, wherein the disorderis an ocular disorder.

110. The method of paragraph 109, wherein the ocular disorder is aMendelian-heritable retinal dystrophy.

111. The method of paragraph 110, wherein the ocular disorder is LCA,Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy,exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C, age-relatedmacular degeneration, retinitis pigmentosa, stickler syndrome,microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB 2, Ushersyndrome, or Wagner syndrome.

Other embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs: 1. An isolatedcircular DNA vector comprising one or more heterologous genes encoding atherapeutic replacement protein, wherein the DNA vector lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

2. The DNA vector of paragraph 1, wherein the DNA vector comprises aterminal repeat sequence.

3. The DNA vector of paragraph 2, wherein the terminal repeat sequenceis at least 10 bp in length.

4. The DNA vector of any one of paragraphs 1-3, wherein the terminalrepeat sequence is a DD element.

5. The DNA vector of any one of paragraphs 1-4, wherein the DNA vectorlacks an immunogenic bacterial signature.

6. The DNA vector of any one of paragraphs 1-5, wherein the DNA vectorlacks an RNA polymerase arrest site.

7. The DNA vector of any one of paragraphs 1-6, wherein the DNA vectorcomprises a promoter which is substantially devoid of CpG islands.

8. The DNA vector of any one of paragraphs 1-7, wherein the therapeuticreplacement protein is secreted into blood.

9. The DNA vector of any one of paragraphs 1-8, wherein the one or moreheterologous genes comprises a trans-splicing molecule or a portionthereof (e.g., a binding domain).

10. The DNA vector of any one of paragraphs 1-9, wherein the DNA vectoris double stranded.

11. The DNA vector of any one of paragraphs 1-10, wherein the DNA vectoris monomeric.

12. The DNA vector of any one of paragraphs 1-11, wherein the DNA vectoris covalently closed.

13. The DNA vector of any one of paragraphs 1-12, wherein the DNA vectoris supercoiled.

14, The DNA vector of any one of paragraphs 1-13, herein the DNA vectoris covalently closed and supercoiled.

15. The DNA vector of any one of paragraphs 1-14, wherein thetherapeutic replacement protein is indicated for treatment of an oculardisorder.

16. The DNA vector of paragraph 15, wherein the ocular disorder is aretinal dystrophy.

17. The DNA vector of paragraph 16, wherein the retinal dystrophy isselected from the group consisting of leber's congenital amaurosis(LCA), Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy,exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C, age-relatedmacular degeneration, retinitis pigmentosa, stickler syndrome,microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB 2, Ushersyndrome, and Wagner syndrome.

18. The DNA vector of any one of paragraphs 1-14, wherein thetherapeutic replacement protein is indicated for treatment of a bloodcoagulation disorder.

19. The DNA vector of paragraph 18, wherein the blood coagulationdisorder is a hemophilia, von Willebrand's disease, factor XIdeficiency, a fibrinogen disorder, or a vitamin K deficiency.

20. The DNA vector of paragraph 18, wherein the blood coagulationdisorder is characterized by a mutation in a gene encoding forfibrinogen, prothrombin, factor V, factor VII, factor VIII, factor X,factor XI, factor XIII, or an enzyme involved in posttranslationalmodifications thereof, or an enzyme involved in vitamin K metabolism.

21. An isolated circular DNA vector comprising one or more heterologousgenes encoding an antigen-binding protein, wherein the DNA vector lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

22. The DNA vector of paragraph 21, wherein the antigen-binding proteinis an antibody or an antigen-binding fragment thereof.

23. The DNA vector of paragraph 21 or 22, wherein the antigen-bindingprotein binds TNF, LT, IFN-α, IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-21, EMAP-II, GM-CSF, EGF, HER2, HER3, FGF, PDGF, BDNF,CNTF, CSF, G-CSF, NGF, PEDF, TGF, VEGF, gonadotropin, insulin-likegrowth factor, CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40,CD45, CD69, CD80, CD86, CD90, PD-1, PD-L1, amyloid beta, alkalinephosphatase, amyloid protein A, CCR4, folate receptor, mucin 5AC,PCSK-9, phosphatidyl-serine, or sclerostin.

24. The DNA vector of paragraph 22, wherein the antigen-binding proteinis a monoclonal antibody, a bispecific antibody, or an antigen-bindingfragment.

25. The DNA vector of paragraph 1, wherein the DNA vector comprises oneor more unmethylated GATC sequences, one or more unmethylated CCAGGsequences, and/or one or more CCTGG sequences.

26. The DNA vector of paragraph 1, wherein the heterologous gene isgreater than 4.5 Kb in length.

27. An isolated circular DNA vector comprising one or more heterologousgenes encoding an enzyme, a growth factor, a hormone, an interleukin, aninterferon, a cytokine, an anti-apoptosis factor, an anti-diabeticfactor, a coagulation factor, an anti-tumor factor, a liver-secretedprotein, or a neuroprotective factor, wherein the DNA vector lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

28. The DNA vector of paragraph 27, wherein the enzyme is an epigeneticregulator.

29. The DNA vector of paragraph 28, wherein the epigenetic regulator isa histone methyltransferase, a histone demethylase, a histone acetylase,a DNA methyltransferase, or a DNA demethylase.

30. The DNA vector of paragraph 27, wherein the growth factor is BDNF,CNTF, CSF, EGF, FGF, G-SCF, M-CSF, GM-CSF, NGF, PDGF, PEDF, TGF, VEGF,gonadotropin, or an insulin-like growth factor.

31. The DNA vector of paragraph 27, wherein the interleukin is IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, or IL-21.

32. The DNA vector of paragraph 27, wherein the interferon is IFN-α orIFN-γ.

33. The DNA vector of paragraph 27, wherein the coagulation factor isfactor V, factor VII, factor VIII, factor IX, factor X, factor XI,factor XII, factor XIII, or von Willebrand factor.

34. The DNA vector of paragraph 27, wherein the neuroprotective factoris selected from the group consisting of a neurotrophin, Kifap3, Bcl-xl,Crmp1, Chk.beta., CALM2, Caly, NPG11, NPT1, Eef1a1, Dhps, Cd151,Morf412, CTGF, LDH-A, Atl1, NPT2, Ehd3, Cox5b, Tuba1a, gamma-actin,Rpsa, NPG3, NPG4, NPG5, NPG6, NPG7, NPG8, NPG9, and NPG10.

35. The DNA vector of paragraph 34, wherein the neurotrophin is selectedfrom the group consisting of NGF, BDNF, NT-3, NT-4, and CNTF.

36. An isolated circular DNA vector comprising one or more heterologousgenes associated with a disorder selected from the group consisting ofan ocular disorder, a liver disorder, a neurological disorder, an immunedisorder, a cancer, a cardiovascular disorder, a blood coagulationdisorder, a lysosomal storage disorder, or type 2 diabetes, wherein theDNA vector lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

37. The DNA vector of paragraph 36, wherein the disorder is an oculardisorder that is a retinal dystrophy.

38. The DNA vector of paragraph 37, wherein the disorder is aMendelian-heritable retinal dystrophy.

39. The DNA vector of paragraph 38, wherein the Mendelian-heritableretinal dystrophy is selected from the group consisting of leber'scongenital amaurosis (LCA), Stargardt Disease, pseudoxanthoma elasticum,rod cone dystrophy, exudative vitreoretinopathy, Joubert Syndrome,CSNB-1C, age-related macular degeneration, retinitis pigmentosa,stickler syndrome, microcephaly and chorioretinopathy, retinitispigmentosa, CSNB 2, Usher syndrome, and Wagner syndrome.

40. The DNA vector of paragraph 36, wherein the disorder is a cancer andthe heterologous gene is CD40, CD40L, CD46, XCL1, MDA-7, IL-12, IL-24,or OPCML (opioid binding protein/cell adhesion molecule).

41. The DNA vector of paragraph 36, wherein the disorder is a cancer andthe heterologous gene is a tumor suppressor gene.

42. The DNA vector of paragraph 41, wherein the tumor suppressor gene isa gene encoding an intracellular protein.

43. The DNA vector of paragraph 41, wherein the tumor suppressor gene isa gene encoding a receptor or signal transducer for a secreted hormoneor developmental signal that inhibits cell proliferation.

44. The DNA vector of paragraph 41, wherein the tumor suppressor gene isa gene that encodes a checkpoint control protein.

45. The DNA vector of paragraph 41, wherein the tumor suppressor gene isa gene that encodes a pro-apoptotic protein.

46. The DNA vector of paragraph 41, wherein the tumor suppressor gene isa gene that encodes a DNA repair protein.

47. The DNA vector of paragraph 36, wherein the coagulation disorder isa hemophilia, von Willebrand's disease, factor XI deficiency, afibrinogen disorder, or a vitamin K deficiency.

48. The DNA vector of paragraph 36, wherein the coagulation disorder ischaracterized by a mutation in a gene encoding for fibrinogen,prothrombin, factor V, factor VII, factor VIII, factor X, factor XI,factor XIII, or an enzyme involved in posttranslational modificationsthereof, or an enzyme involved in vitamin K metabolism.

49. The DNA vector of paragraph 36 or 48, wherein the coagulationdisorder is characterized by a mutation in FGA, FGB, FGG, F2, F5, F7,F10, F11, F13A, F13B, LMAN1, MCFD2, GGCX, or VKORC1.

50. The DNA vector of paragraph 36, wherein the neurological disorder isa neurodegenerative disease.

51. The DNA vector of paragraph 50, wherein the neurodegenerativedisease is selected from the group consisting of Alzheimer's disease,Parkinson's disease, and multiple sclerosis.

52. The DNA vector of paragraph 50, wherein the neurodegenerativedisease is an autoimmune disease of the central nervous system (CNS).

53. The DNA vector of paragraph 52, wherein the autoimmune disease ofthe CNS is multiple sclerosis, encephalomyelitis, a paraneoplasticsyndrome, autoimmune inner ear disease, or opsoclonus myoclonussyndrome.

54. The DNA vector of paragraph 36, wherein the neurological disorder isa cerebral infarction, spinal cord injury, central nervous systemdisorder, a neuropsychiatric disorder, or a channelopathy.

55. The DNA vector of paragraph 54, wherein the channelopathy isepilepsy or migraine.

56. The DNA vector of paragraph 36, wherein the neurological disorder isan anxiety disorder, a mood disorder, a childhood disorder, a cognitivedisorder, schizophrenia, a substance related disorder, or an eatingdisorder.

57. The DNA vector of paragraph 36, wherein the neurological disorder isa symptom of a cerebral infarction, stroke, traumatic brain injury, orspinal cord injury.

58. The DNA vector of paragraph 36, wherein the lysosomal storagedisorder is selected from the group consisting of Tay-Sachs disease,Gaucher disease, Fabry disease, Pompe disease, Niemann-Pick disease, andmucopolysaccharidosis (MPS).

59. The DNA vector of paragraph 36, wherein the cardiovascular disorderis a degenerative heart disease, a coronary artery disease, an ischemia,angina pectoris, an acute coronary syndrome, a peripheral vasculardisease, a peripheral arterial disease, a cerebrovascular disease, oratherosclerosis.

60. The DNA vector of paragraph 36, wherein the cardiovascular disorderis a degenerative heart disease selected from the group consisting of anischemic cardiomyopathy, a conduction disease, and a congenital defect.

61. The DNA vector of paragraph 36, wherein the immune disorder is anautoimmune disorder.

62. The DNA vector of paragraph 61, wherein the autoimmune disorder istype 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus,encephalomyelitis, a paraneoplastic syndrome, autoimmune inner eardisease, or opsoclonus myoclonus syndrome, autoimmune hepatitis,uveitis, autoimmune retinopathy, neuromyelitis optica, psoriaticarthritis, psoriasis, myasthenia gravis, chronic Lyme disease, celiacdisease, chronic inflammatory demyelinating polyneuropathy, peripheralneuropathy, fibromyalgia, Hashimoto's thyroiditis, ulcerative colitis,or Kawasaki disease.

63. The DNA vector of paragraph 36, wherein the disease is a liverdisease selected from the group consisting of hepatitis, Alagillesyndrome, biliary atresia, liver cancer, cirrhosis, a cystic disease,Caroli's syndrome, congenital hepatic fibrosis, fatty liver,galactosemia, primary sclerosing cholangitis, tyrosinemia, glycogenstorage disease, Wilson's disease, and an endocrine deficiency.

64. The DNA vector of paragraph 63, wherein the liver disease is a livercancer selected from the group consisting of a hepatocellularhyperplasia, a hepatocellular adenomas, a focal nodular hyperplasia, ora hepatocellular carcinoma.

65. The DNA vector of paragraph 36, wherein the cancer is a blood canceror a solid tissue cancer.

66. The DNA vector of paragraph 65, wherein the blood cancer is acutelymphoblastic leukemia, acute myeloblastic leukemia, chromic myelogenousleukemia, Hodgkin's disease, multiple myeloma, and non-Hodgkin'slymphoma.

67. The DNA vector of paragraph 65, wherein the solid tissue cancer is aliver cancer, kidney cancer, a breast cancer, a prostate cancer, agastric cancer, an esophageal cancer, a stomach cancer, an intestinalcancer, a colorectal cancer, a bladder cancer, a head and neck cancer, askin cancer, or a brain cancer.

68, The DNA vector of paragraph 36, wherein the heterologous geneencodes a transcription factor.

69. The DNA vector of paragraph 36, wherein the transcription factor isTSHZ2, HOXA2, MEIS2, HOXA3, HAND2, HOXA5, TBX18, PEG3, GLI2, CLOCK,HNF4A, VHL/HIF, WT-1, GSK-3, SPINT2, SMAD2, SMAD3, or SMAD4.

70. The DNA vector of any one of paragraphs 1-69, wherein the disorderis a recessively inherited disorder.

71. The DNA vector of any one of paragraphs 1-70, wherein theheterologous gene is expressible in a target cell selected from thegroup consisting of a liver cell, a retinal cell, a stem cell, a neuralcell, a muscle cell, or a blood cell.

72. The DNA vector of any one of paragraphs 1-71, wherein theheterologous gene is expressible in a post-mitotic target cell.

73. The DNA vector of paragraph 71 or 72, wherein the target cell is aneural cell selected from the group consisting of a neuron, anastrocyte, an oligodendrocyte, and a Schwann cell.

74. The DNA vector of any one of paragraphs 1-73, wherein thetherapeutic protein is secreted into blood.

75. The DNA vector of any one of paragraphs 1-74, wherein the DNA vectorcomprises a promoter sequence upstream of the one or more heterologousgenes.

76. The DNA vector of any one of paragraphs 1-75, wherein the DNA vectorcomprises a polyadenylation site downstream of the one or moreheterologous genes.

77. The DNA vector of any one of paragraphs 1-76, wherein the one ormore heterologous genes comprises a trans-splicing molecule or a portionthereof (e.g., a binding domain).

78. The DNA vector of any one of paragraphs 1-77, wherein the DNA vectorcomprises a terminal repeat sequence.

79. An isolated circular DNA vector comprising one or more therapeuticnucleic acids, wherein the DNA vector:

-   -   (a) lacks an origin of replication and/or a drug resistance        gene;    -   (b) lacks a recombination site; and    -   (c) comprises a terminal repeat sequence.

80. The DNA vector of 79 66, wherein the therapeutic nucleic acid is ansiRNA, shRNA, miRNA, or CRISPRi molecule.

81. The DNA vector of paragraph 79 or 80, wherein the terminal repeatsequence is at least 10 bp in length.

82. The DNA vector of any one of paragraphs 79-81, wherein the terminalrepeat sequence is a DD element.

83. The DNA vector of any one of paragraphs 1-82, wherein the DNA vectorcomprises a suicide gene.

84. The DNA vector of any one of paragraphs 1-83, wherein the DNA vectorlacks bacterial plasmid DNA.

85. The DNA vector of any one of paragraphs 1-79, wherein the DNA vectorcomprises one or more unmethylated GATC sequences, one or moreunmethylated CCAGG sequences, and/or one or more CCTGG sequences.

86. The DNA vector of any one of paragraph 1-85, wherein the DNA vector:

-   -   (a) lacks an immunogenic bacterial signature;    -   (b) lacks an RNA polymerase arrest site; and/or    -   (c) is substantially devoid of CpG islands.

87. The DNA vector of any one of paragraphs 1-86, wherein theheterologous gene is greater than 4.5 Kb in length.

88. The DNA vector of any one of paragraphs 1-87, wherein the DNA vectoris double stranded.

89. The DNA vector of any one of paragraphs 1-88, wherein the DNA vectoris monomeric.

90. The DNA vector of any one of paragraphs 1-89, wherein the DNA vectoris supercoiled.

91. A composition comprising a plurality of the DNA vectors of any oneof paragraphs 1-90.

92. The composition of paragraph 91, wherein at least 50% of theplurality of the DNA vectors comprises one or more unmethylated GATCsequences, one or more unmethylated CCAGG sequences, and/or one or moreCCTGG sequences.

93. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises a heterologous gene encoding a therapeutic protein, whereinthe DNA molecule lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

94. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises one or more heterologous genes encoding an antigen-bindingprotein, wherein the DNA molecule lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

95. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises one or more heterologous genes encoding an enzyme, a growthfactor, a hormone, an interleukin, an interferon, a cytokine, ananti-apoptosis factor, an anti-diabetic factor, a coagulation factor, ananti-tumor factor, a liver-secreted protein, or a neuroprotectivefactor, wherein the DNA molecule lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

96. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises one or more heterologous genes associated with a disorderselected from the group consisting of an ocular disorder, a liverdisorder, a neurological disorder, an immune disorder, a cancer, acardiovascular disorder, a blood coagulation disorder, a lysosomalstorage disorder, or type 2 diabetes, wherein the DNA molecule lacks:

-   -   (a) an origin of replication and/or a drug resistance gene; and    -   (b) a recombination site.

97. An isolated linear DNA molecule comprising a plurality of identicalamplicons, wherein each of the plurality of identical ampliconscomprises one or more therapeutic nucleic acids, wherein the DNAmolecule:

-   -   (a) lacks an origin of replication and/or a drug resistance        gene;    -   (b) lacks a recombination site; and    -   (c) comprises a terminal repeat sequence.

98. The isolated linear DNA molecule of any one of paragraphs 93-97,comprising a restriction enzyme site.

99. The isolated linear DNA molecule of paragraph 98, wherein therestriction enzyme site is positioned between the heterologous gene anda terminal repeat sequence.

100. A method of producing the isolated DNA vector of any one ofparagraphs 1-90, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene;    -   (ii) amplifying the AAV genome using polymerase-mediated        rolling-circle amplification to generate a linear concatamer;    -   (iii) digesting the concatamer using a restriction enzyme to        generate multiple AAV genomes; and    -   (iv) allowing each of the multiple AAV genomes to self-ligate to        produce an isolated DNA vector comprising the heterologous gene.

101. The method of paragraph 100, wherein the AAV genome comprises aterminal repeat sequence.

102. The method of paragraph 100 or 101, further comprising columnpurifying the isolated DNA vector comprising the heterologous gene topurify supercoiled DNA from the isolated DNA vector.

103. A method of producing the isolated DNA vector of any one ofparagraphs 1-90, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene and a terminal repeat sequence;    -   (ii) amplifying the AAV genome using a first polymerase-mediated        rolling-circle amplification to generate a first linear        concatamer;    -   (iii) digesting the first linear concatamer using a restriction        enzyme to generate a first AAV genome;    -   (iv) cloning the first AAV genome into a plasmid vector;    -   (v) identifying a plasmid clone comprising a terminal repeat        sequence;    -   (vi) digesting the plasmid clone comprising the terminal repeat        sequence to generate a second AAV genome;    -   (vii) allowing the second AAV genome to self-ligate to produce a        circular DNA template;    -   (viii) amplifying the circular DNA template using second        polymerase-mediated rolling-circle amplification to generate a        second linear concatamer;    -   (ix) digesting the second linear concatamer using a restriction        enzyme to generate a third AAV genome; and    -   (x) allowing the third AAV genome to self-ligate to produce an        isolated DNA vector comprising the heterologous gene and the        terminal repeat sequence.

104. The method of any one of paragraphs 100-103, wherein thepolymerase-mediated rolling-circle amplification is isothermalrolling-circle amplification.

105. The method of any one of paragraphs 100-104, wherein the polymeraseis Phi29 DNA polymerase.

106. A cell-free method of producing the isolated DNA vector of any oneof paragraphs 1-71, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene;    -   (ii) amplifying the AAV genome using polymerase-mediated        rolling-circle amplification to generate a linear concatamer;    -   (iii) digesting the concatamer using a restriction enzyme to        generate an AAV genome; and    -   (iv) allowing the AAV genome to self-ligate to produce the        isolated DNA vector comprising the heterologous gene.

107. The method of paragraph 106, further comprising column purifyingthe isolated DNA vector comprising the heterologous gene to purifysupercoiled DNA from the isolated DNA vector.

108. The method of paragraph 106 or 107, wherein the polymerase-mediatedrolling-circle amplification is isothermal rolling-circle amplification.

109. The method of any one of paragraphs 106-108, wherein the polymeraseis Phi29 DNA polymerase.

110. A method of producing the isolated DNA vector of any one ofparagraphs 1-90, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene and a DD element;    -   (ii) amplifying the AAV genome using polymerase-mediated        rolling-circle amplification to generate a linear concatamer;    -   (iii) digesting the concatamer using a restriction enzyme to        generate multiple AAV genomes; and    -   (iv) allowing each of the multiple AAV genomes to self-ligate to        produce an isolated DNA vector comprising the heterologous gene        and the DD element.

111. A method of producing the isolated DNA vector of any one ofparagraphs 1-90, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene and a DD element;    -   (ii) amplifying the AAV genome using a first polymerase-mediated        rolling-circle amplification to generate a first linear        concatamer;    -   (iii) digesting the first linear concatamer using a restriction        enzyme to generate a first AAV genome;    -   (iv) cloning the first AAV genome into a plasmid vector;    -   (v) identifying a plasmid clone comprising a DD element;    -   (vi) digesting the plasmid clone comprising the DD element to        generate a second AAV genome;    -   (vii) allowing the second AAV genome to self-ligate to produce a        circular DNA template;    -   (viii) amplifying the circular DNA template using second        polymerase-mediated rolling-circle amplification to generate a        second linear concatamer;    -   (ix) digesting the second linear concatamer using a restriction        enzyme to generate a third AAV genome; and    -   (x) allowing the third AAV genome to self-ligate to produce an        isolated DNA vector comprising the heterologous gene and the DD        element.

112. The method of paragraph 110 or 111, wherein the polymerase-mediatedrolling-circle amplification is isothermal rolling-circle amplification.

113. The method of any one of paragraphs 110-112, wherein the polymeraseis Phi29 DNA polymerase.

114. A cell-free method of producing the isolated DNA vector of any oneof paragraphs 1-90, the method comprising:

-   -   (i) providing a sample comprising a circular DNA vector        comprising an AAV genome, wherein the AAV genome comprises the        heterologous gene and a DD element;    -   (ii) amplifying the AAV genome using polymerase-mediated        rolling-circle amplification to generate a linear concatamer;    -   (iii) digesting the concatamer using a restriction enzyme to        generate an AAV genome; and    -   (iv) allowing the AAV genome to self-ligate to produce a        therapeutic DNA vector comprising the heterologous gene and the        DD element.

115. The method of paragraph 114, wherein the polymerase-mediatedrolling-circle amplification is isothermal rolling-circle amplification.

116. The method of paragraph 114 or 115, wherein the polymerase is Phi29DNA polymerase.

117. A method of inducing episomal expression of a heterologous gene ina subject in need thereof, the method comprising administering to thesubject the isolated DNA vector of any one of paragraphs 1-90 or thecomposition of paragraph 91 or 92.

118. A method of treating a disorder in a subject, the method comprisingadministering to the subject the isolated DNA vector of any one ofparagraphs 1-90 or the composition of paragraph 91 or 92 in atherapeutically effective amount.

119. The method of paragraph 117 or 118, wherein the isolated DNA vectoror the composition is administered repeatedly.

120. The method of paragraph 117 or 118, wherein the isolated DNA vectoror the composition is administered systemically.

121. The method of any one of paragraphs 117-120, wherein the isolatedDNA vector or the composition is administered intravenously.

122. The method of any one of paragraphs 117-120, wherein the isolatedDNA vector or the composition is administered locally.

123. The method of paragraph 122, wherein the isolated DNA vector or thecomposition is administered intravitreally.

124. The method of any one of paragraphs 117-123, wherein the disorderis an ocular disorder.

125. The method of paragraph 124, wherein the ocular disorder is aMendelian-heritable retinal dystrophy.

126. The method of paragraph 124, wherein the ocular disorder is LCA,Stargardt Disease, pseudoxanthoma elasticum, rod cone dystrophy,exudative vitreoretinopathy, Joubert Syndrome, CSNB-1C, age-relatedmacular degeneration, retinitis pigmentosa, stickler syndrome,microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB 2, Ushersyndrome, or Wagner syndrome.

127. The method of any one of paragraphs 117-126, wherein the isolatedDNA vector or the composition is administered systemically.

128. The method of paragraph 127, wherein the disorder is a coagulationdisorder.

129. The method of paragraph 128, wherein the coagulation disorder ishemophilia, von Willebrand's disease, factor XI deficiency, a fibrinogendisorder, or a vitamin K deficiency.

Other Embodiments

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and can be applied to theessential features hereinbefore set forth, and follows in the scope ofthe claims.

Other embodiments are within the claims.

The invention claimed is:
 1. A pharmaceutical composition for deliveryof a gene in a subject, wherein the pharmaceutical composition comprisesa synthetic circular DNA vector comprising a promoter operably linked tothe gene, wherein the synthetic circular DNA vector lacks an origin ofreplication, a drug resistance gene, and a site-specific recombinationrecognition site, wherein the promoter is capable of inducing expressionof the gene in the subject.
 2. The pharmaceutical composition of claim1, further comprising a delivery vehicle selected from liposomes,nanoparticles, microparticles, microspheres, lipid particles, vesicles,polyaxamer, and polycationic material, and combinations thereof.
 3. Thepharmaceutical composition of claim 1, wherein the promoter issubstantially devoid of CpG islands.
 4. The pharmaceutical compositionof claim 1, wherein the synthetic circular DNA vector further comprisesa polyadenylation site downstream of the gene.
 5. The pharmaceuticalcomposition of claim 1, wherein the synthetic circular DNA vector issupercoiled.
 6. The pharmaceutical composition of claim 1, wherein 70%to 99.9% of the synthetic circular DNA vector in the pharmaceuticalcomposition is monomeric.
 7. The pharmaceutical composition of claim 1,wherein the pharmaceutical composition is formulated to be administeredintravenously, intramuscularly, intradermally, intracerebrally,intrathecally, intratumorally, intratracheally, intrapleurally, or byinhalation.
 8. The pharmaceutical composition of claim 1, wherein thegene is at least 5 Kb in length.
 9. The pharmaceutical composition ofclaim 1, wherein the synthetic circular DNA vector is substantiallydevoid of CpG islands.
 10. The pharmaceutical composition of claim 1,wherein the pharmaceutical composition does not cause significantincreases of cytokine levels when administered to a subject.
 11. Thepharmaceutical composition of claim 1, wherein the gene encodes apolypeptide.
 12. The pharmaceutical composition of claim 11, wherein thepolypeptide is a replacement polypeptide.
 13. The pharmaceuticalcomposition of claim 1, wherein the gene encodes a therapeutic nucleicacid.
 14. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition comprises a unit dose of the syntheticcircular DNA vector of from 10 μg to 10 mg.
 15. The pharmaceuticalcomposition of claim 1, wherein the pharmaceutical composition issubstantially devoid of an immunogenic component.
 16. The pharmaceuticalcomposition of claim 15, wherein the immunogenic component comprisesendotoxin, bacterial contaminants, flagellin, lipoteichoic acid,peptidoglycan, or a combination thereof.
 17. The pharmaceuticalcomposition of claim 1, wherein the gene is a human gene.